Emulsion chemistry for encapsulated droplets

ABSTRACT

System, including methods, apparatus, compositions, and kits, for making and using a stabilized emulsion. A method of generating a stabilized emulsion is provided. In the method, an aqueous phase may be provided. The aqueous phase may include an effective concentration of one or more skin-forming proteins. An emulsion may be formed. The emulsion may include droplets of a dispersed phase disposed in a continuous phase, with the aqueous phase being the continuous phase or the dispersed phase. The emulsion may be heated to create an interfacial skin between each droplet and the continuous phase, to transform the droplets into capsules.

CROSS-REFERENCES TO PRIORITY APPLICATIONS

This application is based upon and claims the benefit under 35 U.S.C.§119(e) of U.S. Provisional Patent Application Ser. No. 61/309,845,filed Mar. 2, 2010; U.S. Provisional Patent Application Ser. No.61/341,218, filed Mar. 25, 2010; U.S. Provisional Patent ApplicationSer. No. 61/317,635, filed Mar. 25, 2010; U.S. Provisional PatentApplication Ser. No. 61/380,981, filed Sep. 8, 2010; U.S. ProvisionalPatent Application Ser. No. 61/409,106, filed Nov. 1, 2010; U.S.Provisional Patent Application Ser. No. 61/409,473, filed Nov. 2, 2010;U.S. Provisional Patent Application Ser. No. 61/410,769, filed Nov. 5,2010; and U.S. Provisional Patent Application Ser. No. 61/417,241, filedNov. 25, 2010; each of which is incorporated herein by reference in itsentirety for all purposes.

CROSS-REFERENCES TO ADDITIONAL MATERIALS

This application incorporates by reference in their entirety for allpurposes the following materials: U.S. Pat. No. 7,041,481, issued May 9,2006; U.S. patent application Ser. No. 12/862,542, filed Aug. 24, 2010;U.S. Patent Application Publication No. 2010/0173394 A1, published Jul.8, 2010; and Joseph R. Lakowicz, PRINCIPLES OF FLUORESCENCE SPECTROSCOPY(2^(nd) Ed. 1999).

INTRODUCTION

Many biomedical applications rely on high-throughput assays of samplescombined with reagents. For example, in research and clinicalapplications, high-throughput genetic tests using target-specificreagents can provide accurate and precise quantification of nucleicacids for drug discovery, biomarker discovery, and clinical diagnostics,among others.

The trend is toward reduced volumes and detection of more targets.However, mixing smaller volumes can require more complexinstrumentation, which increases cost. Also, assays performed in smallervolumes may tend be less accurate. Accordingly, improved technology isneeded to permit testing more combinations of samples and reagents, at ahigher speed, a lower cost, with reduced instrument complexity, and/orwith greater accuracy and precision, among others.

Emulsions hold substantial promise for revolutionizing high-throughputassays. Emulsification techniques can create billions of aqueousdroplets that function as independent reaction chambers for biochemicalreactions. For example, an aqueous sample (e.g., 200 microliters) can bepartitioned into droplets (e.g., four million droplets of 50 picoliterseach) to allow individual sub-components (e.g., cells, nucleic acids,proteins) to be manipulated, processed, and studied discretely in amassively high-throughput manner.

Splitting a sample into droplets offers numerous advantages. Smallreaction volumes (picoliters to nanoliters) can be utilized, allowingearlier detection by increasing reaction rates and forming moreconcentrated products. Also, a much greater number of independentmeasurements (thousands to millions) can be made on the sample, whencompared to conventional bulk volume reactions performed on a micoliterscale. Thus, the sample can be analyzed more accurately (i.e., morerepetitions of the same test) and in greater depth (i.e., a greaternumber of different tests). In addition, small reaction volumes use lessreagent, thereby lowering the cost per test of consumables. Furthermore,microfluidic technology can provide control over processes used forgeneration, mixing, incubation, splitting, sorting, and detection ofdroplets, to attain repeatable droplet-based measurements.

Aqueous droplets can be suspended in oil to create a water-in-oilemulsion (W/O). The emulsion can be stabilized with a surfactant toreduce coalescence of droplets during heating, cooling, and transport,thereby enabling thermal cycling to be performed. Accordingly, emulsionshave been used to perform single-copy amplification of nucleic acidtarget molecules in droplets using the polymerase chain reaction (PCR).

Compartmentalization of single molecules of a nucleic acid target indroplets of an emulsion alleviates problems encountered in amplificationof larger sample volumes. In particular, droplets can promote moreefficient and uniform amplification of targets from samples containingcomplex heterogeneous nucleic acid populations, because samplecomplexity in each droplet is reduced. The impact of factors that leadto biasing in bulk amplification, such as amplification efficiency, G+Ccontent, and amplicon annealing, can be minimized by dropletcompartmentalization. Unbiased amplification can be critical indetection of rare species, such as pathogens or cancer cells, thepresence of which could be masked by a high concentration of backgroundspecies in complex clinical samples.

The accuracy and reproducibility of droplet-based assays often relies ondroplets having a uniform, stable size. However, maintaining theintegrity of droplets can present a challenge. Manipulation andprocessing of droplets can cause the droplets to break, coalesce, orboth, which can change an emulsion with a uniform size of droplets (amonodisperse emulsion) to one with a wide range of droplets (apolydisperse emulsion). For example, emulsions can become unstable asthe packing density of droplets is increased, because droplet proximityenables coalescence. This instability limits the ability to storedroplets. Also, the tendency of droplets to coalesce at a high packingdensity restricts the options for batch processing of droplets in a bulkphase. The tendency of droplets both to coalesce and break isexacerbated by higher temperatures and particularly the repetitivecycles of heating and cooling that are utilized for PCR amplification ofa nucleic acid target in droplets. In addition, fluidic manipulation candamage droplets. Droplets may be induced to coalesce by an electricfield (“electro-coalescence”), which can be created by a static chargeon a surface. Accordingly, droplets may be induced to coalesce duringfluidic manipulation, such as in a flow channel, or during aspirationinto or dispensing from a pipet tip, among others. Furthermore, emulsiondroplets tend to be susceptible to breakage when subjected to shear,such as when flowing in a channel and/or when there is a sudden changein direction of flow. For quantitative assays, droplet aggregation,coalescence, and breakage can all introduce large errors to make theassays inaccurate and unreliable.

New systems are needed to make and use emulsions having droplets thatare more stable to storage, thermal cycling, a high packing density,and/or fluidic manipulation.

SUMMARY

The present disclosure provides a system, including methods, apparatus,compositions, and kits, for making and using a stabilized emulsion. Amethod of generating a stabilized emulsion is provided. In the method,an aqueous phase may be provided. The aqueous phase may include aneffective concentration of one or more skin-forming proteins. Anemulsion may be formed. The emulsion may include droplets of a dispersedphase disposed in a continuous phase, with the aqueous phase being thecontinuous phase or the dispersed phase. The emulsion may be heated tocreate an interfacial skin between each droplet and the continuousphase, to transform the droplets into capsules.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram illustrating exemplary formation of skinsto encapsulate droplets of an emulsion, in accordance with aspects ofthe present disclosure.

FIG. 2 is a flowchart illustrating an exemplary method of forming astabilized emulsion including droplets encapsulated by a skin and ofusing the encapsulated droplets to perform an assay, in accordance withaspects of the present disclosure.

FIG. 3 is a schematic illustration of an exemplary approach of removinga continuous phase selectively from an emulsion to increase the volumefraction of the dispersed phase, in accordance with aspects of thepresent disclosure.

FIG. 4 is a schematic illustration of an exemplary approach of coveringa primary emulsion with an overlay emulsion, in accordance with aspectsof the present disclosure.

FIG. 5 is a schematic illustration of an exemplary approach of coveringan emulsion with an overlay phase that is immiscible with at least thecontinuous phase of the emulsion, in accordance with aspects of thepresent disclosure.

FIGS. 6A and 6B are a pair of micrographs of capsules that have beenexposed to a spacing fluid composed of an oil phase lacking thesurfactant that was present during droplet generation (FIG. 6A) orcontaining the surfactant (FIG. 6B), in accordance with aspects of thepresent disclosure.

FIGS. 7A-7D are a set of micrographs of capsules formed as in FIGS. 6Aand 6B and exposed to the same spacing fluid as in FIG. 6B but viewed athigher magnification, in accordance with aspects of the presentdisclosure.

DETAILED DESCRIPTION

The present disclosure provides an emulsion chemistry for a system,including methods, apparatus, compositions, and kits, for making andusing droplets encapsulated by a skin. The skin-encapsulated droplets,or capsules, may be resistant to coalescence, aggregation, and breakageover a wide range of thermal and mechanical processing conditions. Thecapsules may be used to provide more stable encapsulation of samples oranalytes, such as nucleic acids, proteins, cells, or the like, and maybe used in a wide range of biomedical applications, such as assays, drugand/or vaccine delivery, housing biomolecular libraries, clinicalimaging applications, and the like.

A method of generating a stabilized emulsion is provided. In the method,an aqueous phase may be provided, which includes an effectiveconcentration of one or more skin-forming proteins. An emulsion also maybe formed, with the emulsion including droplets of the aqueous phasedisposed in a nonaqueous continuous phase. Alternatively, an emulsionmay be formed with the emulsion including droplets of the nonaqueousphase disposed in an aqueous continuous phase. Accordingly, the emulsionmay be an oil-in-water emulsion or a water-in-oil emulsion, amongothers. The emulsion may be heated to create an interfacial skin betweeneach droplet and the continuous phase, to transform the droplets intocapsules.

The aqueous phase provided may include the skin-forming proteins and atleast one surfactant. The protein(s) may be present at a concentrationof at least about 0.01%, 0.03%, or 0.1%, by weight, among others. Insome cases, although the skin may form at a concentration of 0.01%, theskin may not be amplification-compatible unless formed at a higherconcentration of skin-forming protein, such as at least about 0.03%. Anamplification-compatible skin (which may be termed a PCR-compatibleskin) permits amplification, such as by PCR, of a nucleic acid target.In other words, the skin does not inhibit amplification enough, if atall, to prevent the amplification reaction from occurring efficiently.In any event, the skin-forming protein(s) may be present at aconcentration of about 0.01% to 10%, 0.01% to 3%, 0.01% to 1%, 0.03% to10%, 0.03% to 3%, 0.03% to 1%, 0.05% to 2%, or 0.1% to 1% by weight,among others. The protein(s) may, for example, be selected from thegroup consisting of albumin (e.g., bovine serum albumin (BSA)), gelatin,globulin (e.g., beta-lactoglobulin), and casein, among others. The skinmay be a proteinaceous (protein-containing) skin composed at leastsubstantially of the skin-forming protein(s). Alternatively, or inaddition, the skin may not form substantially when the protein(s) isomitted from the aqueous phase (everything else being equal). In otherwords, the protein(s) may be required for skin formation. The surfactantmay, for example, include a block copolymer of polypropylene oxide andpolyethylene oxide.

A nonaqueous phase may be provided and the emulsion may be formed withthe nonaqueous phase as a continuous phase (or a dispersed phase). Thenonaqueous phase may be an organic or oil phase including at least onefluorinated oil and a fluorinated surfactant (e.g., a fluorinatedpolyether and/or a fluorinated alcohol, among others).

A method of emulsion preparation is provided. In the method, aqueousdroplets may be generated in a continuous phase that includes afluorinated oil and a fluorinated surfactant. The droplets may betransformed to capsules each including an aqueous phase encapsulated bya proteinaceous, interfacial skin. A spacing fluid may be added to thecontinuous phase, with the spacing fluid being miscible with thecontinuous phase and having a different composition than the continuousphase.

A method of performing an assay is provided. In the method, an aqueousphase including a sample and an effective concentration of one or moreskin-forming proteins may be provided. An emulsion also may be formed,with the emulsion including droplets of the aqueous phase disposed in anonaqueous continuous phase. The emulsion may be heated (e.g., to atemperature above about 50° C., 55° C. or 90° C.), to create aninterfacial skin between each droplet and the continuous phase, totransform the droplets into capsules. In some embodiments, the emulsionmay be thermally cycled to promote amplification of one or more nucleicacid targets in the capsules. Assay data related to the sample may becollected from the capsules. The assay data may be processed todetermine an aspect of the sample, such as a concentration of an analyte(e.g., one or more nucleic acid targets) in the sample.

Another method of performing an assay is provided. An aqueous phase maybe provided that includes an effective concentration of one or moreskin-forming proteins. An oil phase may be provided that includes atleast one fluorinated oil and a fluorinated surfactant. An emulsion maybe formed that includes droplets of the aqueous phase disposed in theoil phase, or vice versa. The droplets may be transformed into capsulesby creating an interfacial skin between each droplet and the oil phase(or aqueous phase). The capsules may be thermally cycled to amplify anucleic acid target in the capsules. Amplification data may be collectedfrom the capsules.

A composition for generating a stabilized emulsion is provided. Thecomposition may comprise a continuous phase including a fluorinated oiland at least one fluorinated surfactant. The composition also maycomprise a plurality of aqueous droplets disposed in the continuousphase and including an effective concentration of a skin-formingprotein. Heating the emulsion above a threshold temperature may createan interfacial skin between each droplet and the continuous phase, totransform the droplets into capsules.

A stabilized emulsion is provided. The emulsion may comprise acontinuous phase including a fluorinated oil and at least onefluorinated surfactant. The emulsion also may comprise a plurality ofcapsules disposed in the continuous phase, with each capsule including aproteinaceous, interfacial skin encapsulating an aqueous phase.

An assay kit is provided. The assay kit may include an aqueous phaseincluding an effective concentration of one or more skin-formingproteins and a nonaqueous continuous phase including a fluorinated oiland at least one fluorinated surfactant. The assay kit also may includea droplet generator capable of forming an emulsion including droplets ofthe aqueous phase disposed in the nonaqueous continuous phase. Heatingthe emulsion above a threshold temperature may create an interfacialskin between each droplet and the continuous phase, to transform thedroplets into capsules.

The present disclosure provides methods for preparing capsules ofaqueous phases, including aqueous phases suitable for sample analysis,and the capsules prepared thereby. These capsules may be particularlyuseful for small volume PCR analysis. The disclosed methods may involveseparating samples, such as clinical or environmental samples, into manysmall capsules containing an analyte of interest. For example, eachcapsule may contain less than about one copy of a nucleic acid target(DNA or RNA). The nucleic acid or other analyte in these capsules may bereacted, detected, and/or analyzed, using any suitable technique(s). Thepreparation, reaction, detection, and/or analysis of the disclosedcapsules may be performed in series and/or in parallel, alone, or incombination with other processes. The present disclosure emphasizes, butis not limited to, capsules suitable for performing capsule-basedamplification assays.

Further aspects of the emulsion chemistry and a system that uses theemulsion chemistry are described in the following sections, including:(I) definitions, (II) system overview, (III) aqueous phase, (IV)nonaqueous phase, (V) formation of emulsions, (VI) droplettransformation, (VII) capsules, (VIII) spacing fluid, (IX) capsule anddata processing, and (X) examples.

I. DEFINITIONS

Technical terms used in this disclosure have the meanings that arecommonly recognized by those skilled in the art. However, the followingterms may have additional meanings, as described below.

Assay—a procedure that incorporates one or more reactions, and that isused to characterize a sample of interest. Such characterization may beobtained by virtue of one or more signal(s), value(s), data, and/orresult(s) obtained from the procedure(s) and/or reaction(s). An assaymay be performed using at least one “assay mixture” which is acomposition from which one or more test signals are detected, before,during, and/or after processing of the composition to permit a reaction,if any, to occur. A test or assay may determine a presence (e.g.,concentration) or activity, among others, of one or more analytes in asample.

Reaction—a chemical reaction, a binding interaction, a phenotypicchange, or a combination thereof. An exemplary reaction isenzyme-catalyzed conversion of a substrate to a product and/or bindingof a substrate or product to a binding partner.

Reagent—a compound, set of compounds, and/or composition that iscombined with a sample in order to perform a particular test on thesample. A reagent may be a target-specific reagent, which is any reagentcomposition that confers specificity for detection of a particulartarget or analyte in a test. A reagent optionally may include a chemicalreactant and/or a binding partner for the test. A reagent may, forexample, include at least one nucleic acid, protein (e.g., an enzyme),cell, virus, organelle, macromolecular assembly, a potential drug, alipid, a carbohydrate, an inorganic substance, or any combinationthereof, among others. In exemplary embodiments, the reagent may be anamplification reagent, such as at least one primer or a pair of primersfor amplification of a target, and/or at least one probe to provide anamplification signal.

Nucleic acid—a compound comprising a chain of nucleotide monomers. Anucleic acid may be single-stranded or double-stranded (i.e.,base-paired with another nucleic acid), among others. The chain of anucleic acid may be composed of any suitable number of monomers, such asat least about ten or one hundred, among others. Generally, the lengthof a nucleic acid chain corresponds to its source, with syntheticnucleic acids (e.g., nucleic acid reagents such as primers and probes)typically being shorter and biologically produced nucleic acids (e.g.,nucleic acid analytes) typically being longer.

A nucleic acid can have a natural or artificial structure, or acombination thereof. Nucleic acids with a natural structure, namely,deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), have a backboneof alternating pentose sugar groups and phosphate groups. Each pentosegroup is linked to a nucleobase (e.g., a purine (such as adenine (A) orguanine (T)) or a pyrimidine (such as cytosine (C), thymine (T), oruracil (U))). Nucleic acids with an artificial structure are analogs ofnatural nucleic acids and may, for example, be created by changes to thepentose and/or phosphate groups of the natural backbone. Exemplaryartificial nucleic acids include glycol nucleic acids (GNA), peptidenucleic acids (PNA), locked nucleic acid (LNA), threose nucleic acids(TNA), and the like.

The sequence of a nucleic acid is defined by the order in whichnucleobases are arranged along the backbone. This sequence generallydetermines the ability of the nucleic acid to bind specifically to apartner chain (or to form an intramolecular duplex) by hydrogen bonding.In particular, adenine pairs with thymine (or uracil) and guanine pairswith cytosine. A nucleic acid that can bind to another nucleic acid inan antiparallel fashion by forming a consecutive string ofadenine-thymine and guanine-cytosine base pairs with the other nucleicacid is termed “complementary.”

Replication—a process forming a complementary copy of a nucleic acid ora segment thereof. The nucleic acid and/or segment replicated is atemplate (and/or a target) for replication.

Amplification—a process in which a copy number increases. Amplificationmay be a process in which replication occurs repeatedly over time toform multiple copies of a template. Amplification can produce anexponential or linear increase in the number of copies as amplificationproceeds. Exemplary amplification strategies include polymerase chainreaction (PCR), loop-mediated isothermal amplification (LAMP), rollingcircle replication (RCA), cascade-RCA, nucleic acid based amplification(NASBA), and the like. Also, amplification can utilize a linear orcircular template. Amplification can be performed under any suitabletemperature conditions, such as with thermal cycling or isothermally.Furthermore, amplification can be performed, or tested for itsoccurrence, in an amplification mixture, which is any compositioncapable of amplifying a nucleic acid target, if any, in the mixture. Anamplification mixture can include any combination of at least oneprimer, at least one probe, at least one replication enzyme (e.g., atleast one polymerase, such as at least one DNA and/or RNA polymerase),deoxynucleotide (and/or nucleotide) triphosphates (dNTPs and/or NTPs), amagnesium salt, or any combination thereof, among others. Theamplification mixture may include at least one magnesium-dependentenzyme.

PCR—amplification that relies on repeated cycles of heating and cooling(i.e., thermal cycling) to achieve successive rounds of replication. PCRcan be performed by thermal cycling between two or more temperaturesetpoints, such as a higher denaturation temperature and a lowerannealing/extension temperature, or among three or more temperaturesetpoints, such as a higher denaturation temperature, a lower annealingtemperature, and an intermediate extension temperature, among others.PCR can be performed with a thermostable polymerase, such as Taq DNApolymerase. PCR generally produces an exponential increase in the amountof a product amplicon over successive cycles.

RT-PCR (reverse transcription-PCR)—PCR utilizing a complementary DNAtemplate produced by reverse transcription of RNA. RT-PCR permitsanalysis of an RNA sample by (1) forming complementary DNA copies ofRNA, such as with a reverse transcriptase enzyme, and (2) PCRamplification using the complementary DNA as a template.

Amplicon—a product of an amplification reaction. An amplicon can besingle-stranded or double-stranded, or a combination thereof. Anamplicon corresponds to any suitable segment or the entire length of anucleic acid target.

Primer—a nucleic acid capable of, and/or used for, priming replicationof a nucleic acid template. Thus, a primer is a shorter nucleic acidthat is complementary to a longer template. During replication, theprimer is extended, based on the template sequence, to produce a longernucleic acid that is a complementary copy of the template. A primer maybe DNA, RNA, or an analog thereof (i.e., an artificial nucleic acid),and may have any suitable length, such as at least about 10, 15, or 20nucleotides. Exemplary primers are synthesized chemically. Primers maybe supplied as a pair of primers for amplification of a nucleic acidtarget. The pair of primers may be a sense primer and an antisenseprimer that collectively define the opposing ends (and thus the size) ofa resulting amplicon. In some embodiments, at least one primer may bedescribed as a molecular inversion probe (MIP).

Probe—a nucleic acid connected to a label. A probe may be asequence-specific binding partner for a nucleic acid target and/oramplicon. An exemplary probe includes one or more nucleic acidsconnected to a pair of dyes that collectively exhibit fluorescenceresonance energy transfer (FRET) when proximate one another. The pair ofdyes may respectively provide first and second emitters or an emitter (areporter) and a quencher. Fluorescence emission from the pair of dyeschanges when the dyes are separated from one another, such as bycleavage of the probe (e.g., a Taqman probe) during primer extension, orwhen the probe (e.g., a molecular beacon probe) binds to an amplicon. A“molecular inversion probe” may or may not be connected to a label.

Label—an identifying and/or distinguishing marker or identifierconnected to or incorporated into any entity, such as a molecule,molecular complex, compound, biological particle, or droplet. The labelmay be described as labeling the particular entity to produce a labeledentity. A label may, for example, be a dye that renders an entityoptically detectable or at least more optically detectable. Exemplarydyes used for labeling are fluorescent dyes (fluorophores) andfluorescence quenchers.

Binding partner—a member of a pair of members that bind to one another.Each member may be an atom, molecule, molecular complex, compound,and/or biological particle (a cell, virus, organelle, or the like),among others. Binding partners may bind specifically to one another.Specific binding can be characterized by a dissociation constant of lessthan about 10⁻⁴, 10⁻⁶, 10⁻⁸, or 10⁻¹⁰ M. Exemplary specific bindingpartners include biotin and avidin/streptavidin, a sense nucleic acidand a complementary antisense nucleic acid, a primer and its target, anantibody and a corresponding antigen, a receptor and its ligand, anucleic acid and a protein that recognizes a sequence motif present inthe nucleic acid, and the like.

Fluorinated—including fluorine, typically substituted for hydrogen. Anyof the fluorinated compounds disclosed herein may be polyfluorinated,meaning that such compounds each include many fluorines, such as morethan five or ten fluorines, among others. Any of the fluorinatedcompounds disclosed herein also or alternatively may be perfluorinated,meaning that most or all hydrogens have been replaced with fluorine.

II. SYSTEM OVERVIEW

The system of the present disclosure exploits an emulsion chemistry thatenables formation of skins to encapsulate and stabilize droplets of anemulsion. The droplets may be stabilized against thermal and mechanicalstress, among others, to reduce breakage and coalescence.

FIG. 1 shows a schematic diagram illustrating exemplary formation ofskins around droplets. An emulsion 20 is obtained that includes droplets22 of an aqueous dispersed phase 24 disposed in a nonaqueous continuousphase 26. The droplets may be spaced from one another by any suitableaverage distance to generate any suitable packing density. For example,the droplets may be packed closely together, such as with a packedarrangement having a high packing density. A high packing density is apacked arrangement of droplets in which the collective droplet volume ofthe packed arrangement (i.e., the dispersed phase volume) is at leastabout as great as the interstitial volume of the packed arrangement(i.e., the volume of the continuous phase (or portion thereof) that isdisposed among droplets within the packed arrangement). In other words,in a high packing density, the dispersed phase volume is at least about50% of the sum of the dispersed phase volume and the interstitialvolume. A packed arrangement may be produced by a density differencebetween the dispersed phase and the continuous phase that causes thedroplets to be buoyant or to sink in the continuous phase, in responseto gravity and/or application of a centripetal force. If the dropletsare monodisperse, the high packing density may be provided by asubstantially regular arrangement (a lattice arrangement) of thedroplets.

The aqueous phase may be a skin-forming mixture and may include one ormore skin-forming materials 30, such as at least one skin-formingprotein. For example, at least one skin-forming material may belocalized interfacially, that is, near or at an interface or dropletboundary 32 created between each droplet 22 and continuous phase 26.

The droplets may be transformed to capsules. For example, the droplets,the emulsion, and/or the continuous phase may be heated, indicated at34, to form capsules 36. Each capsule includes an interfacial skin 38formed near or at interface 32, to encapsulate an aqueous phase of eachdroplet 22. The capsules, relative to the progenitor droplets, may bemore stable to various treatments. For example, the capsules may permitlonger storage (such as at about 4° C. to 40° C.) without substantialloss of droplet integrity. Also, the capsules may be more resistant tocoalescence and breakage when heated and/or thermocycled to promotereaction and/or amplification. Further, the capsules may be moreresistant to breakage and/or coalescence produced by an electric fieldor mechanical stress (such as fluidic manipulations). Capsules (and/oran emulsion) resistant to coalescence exhibit less than about 5%, 2%, or1% of the capsules coalescing to form larger capsules/droplets in agiven time period, at a given temperature, and with a given capsulepacking density. In some cases, the capsules may be resistant tocoalescence when incubated at 70° C., 80° C., or 90° C., for at least 1,2, 5, or 10 minutes, with the capsules at a high packing density.Alternatively, or in addition, the capsules may be resistant tocoalescence when stored at 4° C., 20° C., or 37° C. for at least oneweek or one month, with the capsules at a high packing density.Furthermore, the capsules may be more resistant to coalescence whensubjected to an electric field (e.g., from a static charge), and moreresistant to coalescence and breakage when manipulated fluidically, suchas at relatively high flow rates and/or with changes in flow directionor pressure. Moreover, the skin may form a biocompatible interface(e.g., in place of an oil-water interface) that reduces adsorption ofanalytes and/or reagents to the interface from within the droplets.

FIG. 2 illustrates an exemplary method 50 of forming dropletsencapsulated by a skin and of using the encapsulated droplets to performan assay. The method steps presented here may be performed in anysuitable order, in any suitable combination, and may be combined withany other method steps or features described elsewhere in the presentdisclosure or in the documents listed above under Cross-References,which are incorporated herein by reference, particularly U.S.Provisional Patent Application Ser. No. 61/309,845, filed Mar. 2, 2010;U.S. Provisional Patent Application Ser. No. 61/317,635, filed Mar. 25,2010; U.S. Provisional Patent Application Ser. No. 61/380,981, filedSep. 8, 2010; U.S. Provisional Patent Application Ser. No. 61/409,106,filed Nov. 1, 2010; U.S. Provisional Patent Application Ser. No.61/409,473, filed Nov. 2, 2010; U.S. Provisional Patent ApplicationSerial No. 61/410,769, filed Nov. 5, 2010; U.S. Provisional PatentApplication Ser. No. 61/417,241, filed Nov. 25, 2010; and U.S. PatentApplication Publication No. 2010/0173394 A1, published Jul. 8, 2010.

Phases for an emulsion may be provided, indicated at 52. The phasesgenerally include an aqueous phase and a nonaqueous phase that areimmiscible with one another. The phases may be formulated to promoteskin formation when the emulsion is heated. For example, the aqueousphase may include one or more skin-forming components. A skin-formingcomponent is any component of a skin-forming mixture that is necessaryfor skin formation: omitting only the skin-forming component from themixture causes the skin not to form, everything else being equal. Anexemplary skin-forming component is a skin-forming protein. Theskin-forming protein (or proteins) may be present at an effectiveconcentration, which is a concentration sufficient to form a detectableskin when the droplets are appropriately heated or treated otherwise topromote skin formation. The aqueous phase also may include a sample andmay be configured to perform a reaction involving the sample. Inexemplary embodiments, the aqueous phase provides a reaction mixture foramplification of at least one nucleic acid target.

The sample may include nucleic acid. The nucleic acid may, for example,be DNA (e.g., genomic DNA), RNA (e.g., messenger RNA and/or genomicRNA), and/or cDNA (DNA produced by reverse transcription of RNA), amongothers.

An emulsion may be formed, indicated at 54. The emulsion may includedroplets of the aqueous phase disposed in a continuous phase provided bythe nonaqueous phase. The nonaqueous phase may include oil and/or may beformed predominantly by oil, such that the emulsion is a water-in-oilemulsion. In some embodiments, each droplet may be separated from thenonaqueous phase by an interfacial layer that is composed substantiallyof one or more skin-forming components.

The emulsion may be heated to create capsules in which droplets areencapsulated by a skin, indicated at 56. Heating may be performed at atemperature and for a time period sufficient to form the skin, such asto convert an interfacial layer composed of one or more skin-formingcomponents to an interfacial skin. The emulsion may be held by acontainer (e.g., a vial, a chamber, a well of a multi-well plate, etc.)while heated (and/or reacted, see below), or may be disposed in and/orflowing along a channel.

In some embodiments, the emulsion may be heated while held in acontainer (e.g., a well of a multi-well plate) that is sealed with apierce-able sealing member (e.g., a foil, a film, or the like). Thesealing member may be conformable. The sealing member may be piercedwith a tip of a fluid transfer device, such as a pipet tip, a needle, orthe like, to permit removal of the emulsion from the container and/oraddition of fluid (and/or reagent) to the container. Removal of at leasta portion of the emulsion from the container may be performed after skinformation and/or after reaction of the capsules (e.g., amplification ofa nucleic acid target in the capsules), among others.

The capsules may be used immediately or may be stored for any suitabletime period before use (e.g., in some cases, stored for at least oneday, week, or month, among others). The resulting capsules generally aremore stable than the droplets. For example, the capsules may be morestable to shear, and may be stored for extended periods withoutdegradation. The stability of the capsules enables bulk processing andmanipulation that can substantially damage droplets not encapsulated byskin.

In some embodiments, the droplets may not be encapsulated by a skin.Accordingly, any of the steps described in this section, elsewhere inthe present disclosure, or in the documents listed above underCross-References, which are incorporated herein by reference, may beperformed with droplets instead or in addition to capsules.

The phase ratio of the emulsion may be changed, indicated at 58, beforeheating to create capsules (56). Changing the phase ratio is optionaland includes any procedure that substantially increases and/or decreasesthe volume fraction of the aqueous phase in the emulsion. For example,the volume fraction of the aqueous phase may be increased by selectivelyremoving a portion of the continuous phase (relative to the aqueousphase) from the emulsion. In some cases, excess continuous phase may beremoved to produce a high volume fraction of the aqueous phase, such asat least about 50%, among others, in the emulsion. To permit selectiveremoval of droplets or the continuous phase, the emulsion may beformulated with the aqueous and nonaqueous phases having differentdensities, such that the droplets tend to sink or float in the emulsion,to promote sedimentation or creaming, respectively. In exemplaryembodiments, the droplets are buoyant (or sink) in the continuous phase,permitting the emulsion to be concentrated by selectively removingdroplets from a top portion (or bottom portion) of the emulsion and/orselectively removing the continuous phase from a bottom portion (or topportion) of the emulsion. Further aspects of changing the phase ratio ofan emulsion are described below in Example 1 of Section X.

In some embodiments, removing a portion of the continuous phase mayimprove stability of droplets prior to and/or after the transformationto capsules. For some cases, such as when the concentration of at leastone ionic surfactant in the oil phase (continuous phase) is above theCMC (critical micelle concentration), there may be an excess of ionicsurfactant that exists in micelles, and these micelles may compete withthe droplets or capsules through a thermodynamic driving force thatdraws water out of the droplets or capsules, causing them to shrink oreven tear (in the case of the skin-bearing capsules). Accordingly, anyapproach that reduces the amount of excess ionic micelles in thecontinuous phase may improve droplet or capsule stability. Some examplesof steps that can be taken include (1) removal of at least a portion ofexcess continuous phase (in other words, removal of some micelles) priorto transformation of droplets to capsules and/or (2) use of reducedsurfactant concentration in (a) the continuous phase at the time ofemulsion formation (provided a sufficient amount of surfactant ispresent to generate and sustain intact droplets), and/or (b) thecontinuous phase for any capsule spacing or transport fluids (describedin more detail elsewhere in the present disclosure).

Another pre-transformation step that may be effective to reduce loss ofwater from droplets/capsules to the continuous phase is to“pre-saturate” or “pre-hydrate” the continuous phase with water, ratherthan or in addition to removing excess continuous phase to reduce thenumber of micelles. The continuous phase may be exposed to water beforeemulsion formation to achieve pre-hydration. For example, the continuousphase may be overlaid or otherwise disposed in contact with a volume ofwater or an aqueous, pre-hydration solution that more closely resembles(ionic, osmotic balance) the aqueous solution that will be disposed inthe droplets when they are subsequently formed with the pre-hydratedcontinuous phase. The pre-hydration solution can, for example, be thebuffered base for the reaction mixture within the droplets, generallyexcluding any nucleic acids or proteins/enzymes. The skin-formingmaterial and any aqueous phase surfactants also may be excluded from thepre-hydration solution in some cases.

The emulsion optionally may be overlaid, indicated at 60. In someembodiments, where the droplets are buoyant compared to the continuousphase, an overlay may be useful to protect droplets from breakage orother degradation during exposure to air and/or other interfaces (e.g.,the air-emulsion interface during a heating step to form capsules). Anoverlay also may be useful where the droplets sink compared to theinterface, although continuous phase above the droplets may render anoverlay unnecessary. In any event, an overlay may be placed onto theemulsion to cover a top surface of the emulsion, before (or after) theemulsion is heated to create capsules (56). The overlay contacts theemulsion and forms a layer that generally remains above the emulsion.Stated differently, the overlay may completely cover an air-exposed, topsurface of the emulsion, to replace an emulsion-air interface with anemulsion-overlay interface, thereby reducing exposure of the emulsion toair. The overlay may reduce evaporation of a component(s) from either orboth phases of the emulsion, such as evaporation of oil and water fromthe continuous and aqueous phases, respectively. In any event, theoverlay may reduce droplet damage (e.g., breakage) that can occur beforecapsule formation with some formulations, as the emulsion is beingheated, and/or capsule damage (e.g., desiccation/shrinkage) that canoccur near an emulsion-air interface at relatively higher temperatures,such as during thermal cycling for PCR amplification.

The overlay may be fluid or solid when applied, and, if fluid whenapplied, may remain fluid or may solidify. The overlay (or a continuousphase thereof) may have a lower density than the continuous phase of theunderlying emulsion, such that the overlay floats on the primaryemulsion. A fluid overlay may have any suitable composition. Forexample, the fluid overlay may be an overlay emulsion (containingdroplets and/or capsules) or an overlay phase. Further aspects of fluidoverlays are described in Example 2 of Section X.

The emulsion may be reacted, indicated at 62. Generally, reaction of theemulsion involves treating the emulsion to promote individual reactionsin capsules of the emulsion. For example, the emulsion may be heatedand/or thermally cycled to promote amplification of a nucleic acidtarget in the capsules. Accordingly, heating may be part of a thermalcycling process or may be a pre-incubation (e.g., reverse transcription,uracil removal, endo- or exonuclease digestion, etc.) prior to a thermalcycling process. The pre-incubation step or first incubation step in anRT-PCR or PCR protocol may be used to transform droplets to capsules, toform the interfacial skin at a high packing density, at a lower packingdensity, or even with the droplets not packed (such as spaced in fluidunder flow but exposed to heating), in order to further resistcoalescence in subsequent manipulations of the capsules. This may beparticularly useful where subsequent manipulations involve a highpacking density of the capsules.

The droplets may be reacted in parallel, such as in a batch reactionperformed in a container. If reacted in a batch reaction, the capsulesmay be at a high packing density. Alternatively, the droplets may bereacted in parallel or serially as the droplets flow along a channel ofa continuous flow reactor, and/or the droplets may be heated in batch(e.g., at a high packing density) to form the skin, and then flowedthrough the continuous flow reactor (e.g., see U.S. Patent ApplicationPublication No. 2010/0173394 A1, published Jul. 8, 2010, which isincorporated herein by reference), among others.

Signals may be detected from capsules of the emulsion, indicated at 64.Stated differently, assay data related to a sample in the aqueous phasemay be collected from the capsules. The data may relate to at least onereaction involving one or more analytes in the sample. In exemplaryembodiments, the data relates to amplification of a nucleic acid targetin the capsules.

The capsules of the emulsion optionally may be spaced from one another,indicated at 66. Spacing the capsules may be performed one or more timesbefore and/or after reaction of the emulsion and before detection ofsignals from the capsules. Spacing the capsules generally includes anymanipulation that increases the average or local spacing betweencapsules of the emulsion.

The capsule spacing may be increased to facilitate fluidic manipulationof capsules that are in a packed arrangement of high density afterreaction in a container. The packed arrangement may be the result ofcreaming/sedimentation alone or in combination with changing the phaseratio (58), such as by removal of excess continuous phase from theemulsion. In some embodiments, the packed arrangement may form asubstantial lattice of capsules and/or may dispose the capsules in asubstantial crystalline state, if the capsules are monodisperse (and,optionally, if the aqueous fraction is high).

The use of a spacing fluid to facilitate spacing the capsules from oneanother may be based on the volume fraction of the aqueous phase. Theemulsion may have a more fluid consistency if the aqueous phase fractionis lower, such as approximately equal to or less than the continuousphase fraction. In this case, the capsules may (or may not) be dispersedreadily (i.e., spaced farther from one another) without addition of aspacing fluid, such as by agitation of the emulsion. Alternatively, theemulsion may have a less fluid or more “gel-like” consistency, if theaqueous phase fraction is higher, such as higher than the continuousphase fraction. In this case (and/or with the fluid-like emulsion), thecapsules may be dispersed with the aid of a spacing fluid added to theemulsion. In any event, the capsules may be spaced to facilitate flow,such as flow into a conduit of a fluid transport device that picks updroplets from the container, for example, via a tip of the devicedisposed in the emulsion. In some cases, thermally cycling capsulesmakes them “sticky” and more difficult to disperse. Addition of aspacing fluid may facilitate dispersal of sticky capsules.

Capsules also or alternatively may be spaced farther from one another toenable detection of individual droplets. Accordingly, a spacing fluidmay be added to droplets flowing in a channel to a detection region thatis operatively disposed with respect to a detector. The spacing fluidmay be described as a focusing fluid, and may singulate the dropletsbefore they reach the detection region. The singulated droplets maytravel serially through the detection region. Further aspects of spacingdroplets upstream of a detection region are disclosed in the documentslisted above under Cross-References listed above, which are incorporatedherein by reference, particularly U.S. Provisional Patent ApplicationSer. No. 61/317,635, filed Mar. 25, 2010; U.S. Provisional PatentApplication Ser. No. 61/409,106, filed Nov. 1, 2010; U.S. ProvisionalPatent Application Ser. No. 61/409,473, filed Nov. 2, 2010; U.S.Provisional Patent Application Ser. No. 61/410,769, filed Nov. 5, 2010;and U.S. Patent Application Publication No. 2010/0173394 A1, publishedJul. 8, 2010.

The emulsion optionally may be manipulated fluidically, indicated at 68.If the phase ratio is changed (66), such as by selective removal ofdroplets/capsules or the continuous phase, fluidic manipulation may beperformed before, during, and/or after this change.

Fluidic manipulation generally involves moving the emulsion ordroplets/capsules thereof by fluid flow. For example, fluidicmanipulation may include dispersing droplets/capsules disposed in acontainer, introducing droplets/capsules from the container into a flowstream, dispersing droplets/capsules in the flow stream (e.g.,singulating the capsules), and/or driving flow of capsules to adetection region, where a detector may collect data from the capsulesserially or in parallel (e.g., by imaging).

Collected data may be processed, indicated at 70. Data processing mayinclude determining at least one aspect of one or more analytes of oneor more samples included in the aqueous phase of the emulsion. The dataprocessing may include subtracting background, normalizing capsule databased on capsule size, applying a threshold to capsule signals todistinguish positive from negative capsules for the assay, determining aconcentration of an analyte (e.g., a nucleic acid target) in the sample(e.g., based on Poisson statistics), or any combination thereof, amongothers.

The capsules may be used to perform any suitable assay to measure anysuitable characteristic of an analyte. In some embodiments, the analyteis nucleic acid and amplification data from individual capsules may beanalyzed to determine whether or not amplification of one or morenucleic acid targets occurred in individual droplets, in a digitalamplification assay. In other words, the amplification data may beprocessed to provide a digital description of the presence or absence ofeach target in each droplet analyzed. In any event, the amplificationdata may be processed to provide information about any suitable aspectof a sample, such as the presence or absence of at least one singlenucleotide polymorphism, methylation of a target site, copy numbervariation of a target, a rare mutation/target (e.g., a mutationassociated with cancer), fetal aneuploidy, a haplotype, and the like.Further aspects of assays that may be suitable are described in thedocuments listed above Cross-References, which are incorporated hereinby reference, particularly U.S. Patent Application Publication No.2010/0173394 A1, published Jul. 8, 2010; U.S. Provisional PatentApplication Ser. No. 61/380,981, filed Sep. 8, 2010; U.S. ProvisionalPatent Application Ser. No. 61/409,106, filed Nov. 1, 2010; U.S.Provisional Patent Application Ser. No. 61/410,769, filed Nov. 5, 2010;and U.S. Provisional Patent Application Ser. No. 61/417,241, filed Nov.25, 2010.

Further aspects of emulsions, emulsion phases, phase components,generating droplets/forming emulsions, reacting emulsions, detectingsignals, fluidic manipulation, and data processing, among others, thatmay be suitable are described in the documents listed aboveCross-References, which are incorporated herein by reference,particularly U.S. Provisional Patent Application Ser. No. 61/341,218,filed Mar. 25, 2010; U.S. Provisional Patent Application Ser. No.61/317,635, filed Mar. 25, 2010; U.S. Provisional Patent ApplicationSer. No. 61/409,106, filed Nov. 1, 2010; U.S. Provisional PatentApplication Ser. No. 61/409,473, filed Nov. 2, 2010; U.S. ProvisionalPatent Application Ser. No. 61/410,769, filed Nov. 5, 2010; U.S.Provisional Patent Application Ser. No. 61/417,241, filed Nov. 25, 2010;and U.S. Patent Application Publication No. 2010/0173394 A1, publishedJul. 8, 2010.

III. AQUEOUS PHASE

The aqueous phase is substantially and/or predominantly water, but mayincorporate a variety of additional components. The components may besoluble and/or miscible in water, such as one or more salts, bufferingagents, reagents, samples of interest, analytes of interest, and/orwhatever additional components may be necessary for a desiredreaction(s) that may be intended to occur within a formed droplet orcapsule. All such additional components may be selected to be compatiblewith the desired reaction or intended assay. Additionally, the aqueousphase may include one or more skin-forming components.

In some cases, the components may include droplets disposed in theaqueous phase, such as one more simple or compound droplets. Forexample, the aqueous phase may contain one or more oil droplets, whichin turn may (or may not) contain one or more aqueous droplets, and soon. Accordingly, the skin may encapsulate aqueous droplets that fusewith one another within the skin, during and/or after skin formation.Further aspects of forming multiple emulsions and inducing dropletfusion within multiple emulsions are described in U.S. patentapplication Ser. No. 12/862,542, filed Aug. 24, 2010, which isincorporated herein by reference.

Salts and/or Buffers

Any suitable salt or combination of salts may be present in the aqueousphase. Each salt may or may not be a physiologically compatible salt.Exemplary salts for the aqueous phase include any one or combination ofNaCl, KCl, CaCl₂, MgCl₂, and MgSO₄, among others.

Any suitable buffer(s) or buffering agent(s) may be present in theaqueous phase. The buffer or buffering agent may be configured tomaintain the pH of the aqueous phase near or at any suitable pH, such asa pH near or at which a desired reaction or set of reactions occursefficiently (e.g., near an optimum pH for an enzyme activity). In somecases, the pH may, for example, approximate a physiological pH, such asabout 6.5 to 8.5, 7 to 8, or about 7.5 among others. In any event, aparticular buffering agent may be selected that has a pK_(a) relativelyclose to the desired pH to be maintained and that is compatible with thereaction(s) to be performed. For example, the buffering agent may bephysiologically compatible. Exemplary buffering agents that may besuitable include Tris (2-Amino-2-hydroxymethyl-propane-1,3-diol), MES(2-(N-morpholino) ethanesulfonic acid), MOPS(3-morpholinopropane-1-sulfonic acid), HEPES(2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid), and the like.

Reagents

Where the aqueous phase includes one or more reagents, the reagent isunderstood to be a compound, set of compounds, and/or composition thatis combined with a sample of interest in order to perform a particulartest on the sample. A reagent may be a target-specific reagent, which isany reagent composition that confers specificity for reaction with ordetection of a particular target or analyte in a test. A reagentoptionally may include a chemical reactant and/or a binding partner forthe test. A reagent may, for example, include at least one nucleic acid,protein (e.g., an enzyme), cell, virus, organelle, macromolecularassembly, a potential drug, a lipid, a carbohydrate, an inorganicsubstance, or any combination thereof, among others. In exemplaryembodiments, the reagent may be an amplification reagent, such as apolymerase (e.g., a heat-stable polymerase that may or may not require ahot start to activate the polymerase), a reverse transcriptase, aligase, an exonuclease, at least one primer or at least one set ofprimers for amplification of a target, at least one probe to provide anamplification signal for the amplified target, or any combinationthereof, among others. In some cases, the aqueous phase anddroplets/capsules may include a molecular inversion probe (MIP). Furtheraspects of molecular inversion probes and their use indroplet-/capsule-based assays are described in U.S. Provisional PatentApplication Ser. No. 61/380,981, filed Sep. 8, 2010; and U.S.Provisional Patent Application Ser. No. 61/417,241, filed Nov. 25, 2010;each of which is incorporated herein by reference.

Samples

Where the aqueous phase includes a sample, the sample is understood tobe a compound, composition, and/or mixture of interest, from anysuitable source(s). A sample may be the general subject of interest fora test that analyzes an aspect of the sample, such as an aspect relatedto at least one analyte that may be present in the sample. Samples maybe analyzed in their natural state, as collected, and/or in an alteredstate, for example, following storage, preservation, extraction, lysis,dilution, concentration, purification, filtration, mixing with one ormore reagents, partitioning, further processing, or any combinationthereof, among others. Clinical samples may include blood, saliva,urine, stool, sputum, mucous, milk, a fluid aspirate, and/or tissue,among others. Environmental samples may include water, soil, and/or air,among others. Research samples may include cultured cells, primarycells, viruses, small organisms, tissue, a body fluid, or the like.Additional samples may include foodstuffs, weapons components, suspectedcontaminants, and so on.

Analytes

Where the aqueous phase includes an analyte of interest, the analyte isunderstood to be a component(s) or potential component(s) of a samplethat is analyzed in a test. An analyte is a more specific subject ofinterest in a test for which the sample is a more general subject ofinterest. An analyte may, for example, be a nucleic acid, a protein, anenzyme, a cell, a virus, an organelle, a macromolecular assembly, a drugcandidate (and/or potential drug), a lipid, a carbohydrate, an inorganicsubstance, or any combination thereof, among others. An analyte may betested for its concentration, activity, and/or other characteristic in asample. The concentration of an analyte may relate to an absolute orrelative number, binary assessment (e.g., present or absent), or thelike, of the analyte in a sample or in one or more partitions thereof.

Surfactants

A surfactant is a surface-active substance capable of reducing thesurface tension of a liquid in which it is present. A surfactant, whichalso or alternatively may be described as a detergent and/or a wettingagent, may incorporate both a hydrophilic portion and a hydrophobicportion, which may collectively confer a dual hydrophilic-hydrophobiccharacter on the surfactant. A surfactant may, in some cases, becharacterized according to its hydrophilicity relative to itshydrophobicity. The aqueous phase would typically incorporate at leastone hydrophilic surfactant. The aqueous phase may include at least onenonionic surfactant and/or ionic surfactant. In some embodiments, theaqueous phase may include a surfactant that is a block copolymer ofpolypropylene oxide and polyethylene oxide. More particularly, thesurfactant may be a block copolymer of polypropylene oxide andpolyethylene oxide sold under the trade names PLURONIC and TETRONIC(BASF). In some embodiments, the surfactant may be a nonionic blockcopolymer of polypropylene oxide and polyethylene oxide sold under thetrade name PLURONIC F-68. In some embodiments, the surfactant of theaqueous phase may be a water-soluble and/or hydrophilicfluorosurfactant. Exemplary fluorosurfactants for the aqueous phase aresold under the trade name ZONYL (DuPont), such as ZONYL FSNfluorosurfactants. In some cases, the surfactant may include polysorbate20 (sold under the trade name TWEEN-20 by ICI Americas, Inc.). Theconcentration of a particular surfactant or total surfactant present inthe aqueous phase may be selected to stabilize emulsion droplets priorto heating. An exemplary concentration of surfactant for the aqueousphase is about 0.01 to 10%, 0.05 to 5%, 0.1 to 1%, or 0.5% by weight,among others. In some cases, a skin-forming protein may function as asurfactant, although proteins generally are not classified assurfactants for the purposes of the present disclosure.

Skin-Forming Components

The aqueous phase may include one or more skin-forming components. Askin-forming component is any substance that promotes formation of askin near or at the droplet boundary, for example, by serving as astructural element of the skin. Each skin-forming component may have anysuitable distribution with respect to each droplet prior to skinformation. The skin-forming component may be localized selectively nearor at the droplet interface, to form an interface layer, or may bedistributed more uniformly throughout the aqueous phase. If distributedmore uniformly, the skin-forming component may be recruited to theinterface during skin formation.

The skin-forming components may include at least one skin-formingprotein. The protein may be present at an effective concentration, whichis an amount sufficient for detectable skin formation under theappropriate conditions (e.g., heating). Exemplary effectiveconcentrations include at least about 0.01% or 0.03%, 0.03% to 3%, 0.05%to 2%, 0.1% to 1%, or about 0.1% by weight, among others. The proteinmay be described as a “non-specific blocking” or “non-specific binding”protein. The phrase “non-specific blocking” or “non-specific binding” asused herein refers generally to a capability to non-specifically bind tosurfaces, that is, hydrophobic and/or hydrophilic surfaces, sometimeswith the aid of heating. Non-specific blocking/binding proteins aretypically water-soluble proteins, may be relatively large serum or milkproteins (among others), and/or may not interact with any of the othercomponents of the aqueous phase in a specific binding fashion. Exemplarynon-specific blocking/binding proteins that may be suitable as skinforming proteins include albumins (such as a serum albumin (e.g., frombovine (BSA), human, rabbit, goat, sheep or horse, among others)),globulins (e.g., beta-lactoglobulin), casein, and gelatin (e.g., bovineskin gelatin type B), among others.

Additional Additives

The aqueous phase optionally further includes any of a variety ofadditives. The additives, may, for example, be intended to act aspreservatives, enzyme enhancers, enzyme inhibitors, cofactors, and thelike, including, for example, sodium azide, betaine, trehalose, andRNase inhibitors, among others. Other exemplary additives are enzymes,such as a restriction enzyme, a ligase, a reverse transcriptase,Uracil-DNA N-Glycosylase (UNG), and the like.

Treatment Prior to Droplet Formation

The sample and/or the aqueous phase may be treated, prior to dropletgeneration, to facilitate formation of droplets. Treatment may beparticularly suitable with a relatively high concentration and/orrelatively long fragments of nucleic acid in the aqueous phase. Whendroplets are formed under standard conditions, the aqueous phase may besubjected to a rapid decrease in cross sectional area, elongation,followed by separation and formation of the droplet. When DNA, RNA, oranother long-chain polymer is present above certain concentrations, theability to form droplets may be impaired. For example, these polymersmay become entangled with each other in the rapid process of dropletformation, and may not have sufficient time to separate throughdiffusion, there forming a cord that causes the droplets not to formefficiently. The cord may result in jetting, microsatellites, andcoalescence, and other features of poor emulsion formation.Alternatively, or in addition, the polymers may be interacting with thedroplet interface, decreasing surface tension and preventing dropletformation.

In any event, an approach is needed to overcome this effect on dropletformation. One exemplary approach is to slow down the rate of dropletformation so that the droplet has time to pinch off and form. However,this approach reduces the throughput of droplet formation. Anothermechanical solution may be to redesign the droplet generator to forcethe formation of droplets under these high concentration conditions.Another exemplary approach is to fragment the polymer(s) to a smallersize. The polymer may be fragmented by heating the aqueous phase beforeemulsion formation. For example, the aqueous sample may be heated to atleast about 80° C., 90° C., or 95° C. for at least about 1, 2, 5, 10,15, or 30 minutes, among others. Suitable heating may result in theability to form droplets at high DNA concentrations under normalconditions. A further exemplary approach is to fragment DNA in theaqueous phase by digesting the DNA with a restriction enzyme, whichtargets specific sites (or cuts the DNA nonspecifically). As long as thespecific sites are outside of the target of interest, the copy number oftarget measured is preserved in the sample. The digestion may or may notgo to completion. In many cases, only a partial digestion may benecessary to reduce the average DNA fragment size to a level that doesnot impact droplet formation.

Selected Embodiments of the Aqueous Phase

The aqueous phase may be formulated to perform one or more enzymereactions, such as reverse transcription, amplification, restrictionenzyme digestion, ligation, uracil cleavage from carry-over amplicons(to prevent amplification of contaminating targets), any combinationthereof, or the like. For example, the aqueous phase may be formulatedto perform RT-PCR and may include any suitable combination of thecomponents listed in the follow formulations:

Aqueous Phase Formulation 1

-   -   Reaction Buffer (˜50-70 mM [salt]): ˜45-55 mM KCl, ˜10-15 mM        Tris, ˜pH 7.5-8.5)    -   MgCl₂ and/or MgSO₄ (˜1.5-5 mM)    -   BSA or bovine gelatin (˜0.1-1% w/v)    -   Nonionic polyethylene oxide (PEO)/polypropylene oxide (PPO)        block copolymer surfactant (˜0.1-1% w/v)    -   Heat-stable Polymerase (˜0.04 Units/μL)    -   Reverse Transcriptase (−0.04 Units/μL)    -   dNTPs (˜200-400 μM each (dATP, dCTP, dGTP, dTTP) or ˜300-500 μM        each (with dUTP in place of dTTP))    -   UNG (Uracil-DNA N-Glycoslyase) (optional; ˜0.025-0.1 Units/μL)    -   Total nucleic acid (˜pg to ng range/nL, with the target nucleic        acid present at less than ˜10 copies/nL or less than ˜1 copy/nL)    -   Primers (˜0.1-1.0 μM)    -   Probe(s) for Fluorescence Detection (˜0.1-0.25 μM)

Aqueous Phase Formulation 2 (Selected Components)

-   -   KCl (˜50 mM)    -   Tris (˜15 mM, pH 8.0)    -   MgCl₂ (˜3.2 mM)    -   BSA or bovine gelatin (˜0.1% w/v)    -   Pluronic F-68 (surfactant, ˜0.5% w/v)    -   dNTPs (˜200 μM each (dATP, dCTP, dGTP, dTTP))    -   Primers (˜0.5 μM each)    -   Probe(s) for Fluorescence Detection (˜0.25 μM)

IV. NONAQUEOUS PHASE

The nonaqueous phase may serve as a carrier fluid forming a continuousphase that is immiscible with water, or the nonaqueous phase may be adispersed phase. The nonaqueous phase may be referred to as an oil phasecomprising at least one oil, but may include any liquid (or liquefiable)compound or mixture of liquid compounds that is immiscible with water.The oil may be synthetic or naturally occurring. The oil may or may notinclude carbon and/or silicon, and may or may not include hydrogenand/or fluorine. The oil may be lipophilic or lipophobic. In otherwords, the oil may be generally miscible or immiscible with organicsolvents. Exemplary oils may include at least one silicone oil, mineraloil, fluorocarbon oil, vegetable oil, or a combination thereof, amongothers.

In exemplary embodiments, the oil is a fluorinated oil, such as afluorocarbon oil, which may be a perfluorinated organic solvent. Afluorinated oil may be a base (primary) oil or an additive to a baseoil, among others. Exemplary fluorinated oils that may be suitable aresold under the trade name FLUORINERT (3M), including, in particular,FLUORINERT Electronic Liquid FC-3283, FC-40, FC-43, and FC-70. Anotherexample of an appropriate fluorinated oil is sold under the trade nameNOVEC (3M), including NOVEC HFE 7500 Engineered Fluid.

Surfactants

As discussed above with respect to the aqueous phase, a surfactant is asurface-active substance capable of reducing the surface tension of aliquid in which it is dissolved, and may incorporate both a hydrophilicportion and a hydrophobic portion, which may collectively confer a dualhydrophilic-hydrophobic character on the surfactant. In contrast to thesurfactant present in the aqueous phase, the nonaqueous phase wouldtypically incorporate a hydrophobic surfactant. The nonaqueous phase mayinclude one or more surfactants, each of which may be disposed/dissolvedin the nonaqueous phase prior to, during, and/or after capsuleformation. The surfactants may include a nonionic surfactant, an ionicsurfactant (a cationic (positively-charged) or anionic(negatively-charged) surfactant), or both types of surfactant. Exemplaryanionic surfactants that may be suitable include carboxylates,sulphonates, phosphonates, and so on. The one or more surfactants may bepresent, individually or collectively, at any suitable concentration,such as greater than about 0.001% or 0.01%, or about 0.001% to 10%,0.05% to 2%, or 0.05% to 0.5%, among others.

An ionic surfactant (e.g., a negatively-charged surfactant) may bepreferred for capsule formation. The ionic surfactant may promoteattraction for the purpose of assembly of components at the interfacethat can lead to the formation of a skin upon heating. For example,ionic pairing may occur between an ionic surfactant in the continuousphase and a skin-forming protein in the dispersed phase (or vice versaif the continuous phase is aqueous). With the skin-forming protein boundat the interface by the ionic surfactant, application of heat may changethe conformation of the protein (by denaturation) and/or decrease itssolubility in the aqueous phase, which may lead to formation of skin.Alternatively, or in addition, if an ionic or nonionic surfactant isincluded in an oil composition used for emulsion formation, hydrophobicinteractions may recruit a skin-forming protein and/or otherskin-forming material to the interface.

The one or more surfactants present in the nonaqueous phase (or oilphase) may be fluorinated surfactants (e.g., surfactant compounds thatare polyfluorinated and/or perfluorinated). Exemplary fluorinatedsurfactants are fluorinated polyethers, such as carboxylicacid-terminated perfluoropolyethers, carboxylate salts ofperfluoropolyethers, and/or amide or ester derivatives of carboxylicacid-terminated perfluoropolyethers. Exemplary but not exclusiveperfluoropolyethers are commercially available under the trade nameKRYTOX (DuPont), such as KRYTOX-FSH, the ammonium salt of KRYTOX-FSH(“KRYTOX-AS”), or a morpholino derivative of KRYTOX-FSH (“KRYTOX-M”),among others. Other fluorinated polyethers that may be suitable includeat least one polyethylene glycol (PEG) moiety.

A primary surfactant, such as a fluorinated polyether, may be present atany suitable concentration, such as about 0.02% to 10%, or about 1% to4%, by weight. The primary surfactant may be present at either arelatively higher concentration (about 1% or greater by weight) or arelatively lower concentration (less than about 1% by weight, such asabout 0.02 to 0.5% by weight). In some cases, use of the lowerconcentration may enable capsules to be created by heating dropletswithout use of an overlay and without substantial droplet breakage. Theprimary surfactant may (or may not) have a molecular weight of at leastabout 1, 2, or 5 kilodaltons.

The nonaqueous phase may further include one or more additionalsurfactants selected to modify one or more physical properties of aselected oil. For example, an additional surfactant may be used to lowerthe evaporation potential of the selected oil. By lowering theevaporation potential, the additional surfactant may reduce or minimizethe effect of evaporation on droplets at an emulsion-air interface. Inexemplary embodiments, the nonaqueous phase may include a fluorinatedoil, which may be the predominant component, a primary surfactant (e.g.,a fluorinated polyether), and a secondary/additional surfactant, amongothers. The secondary/additional surfactant may be a fluorinated alcoholwith only one (a monoalcohol) or two hydroxyl groups, such asperfluorodecanol or perfluorooctanol, among others. The additionalsurfactant may have a molecular weight of less than about 1000 or 500daltons, may have no more than about 20, 15, or 12 carbons, and may bepresent at a concentration of about 0 to 10%, 0% to 5%, 0 to 2.5%, 0.1%to 2.5%, or 0.001% to 0.5% by weight, among others.

Selected Embodiments of the Nonaqueous Phase

The nonaqueous phase may include any combination of a fluorosurfactant,a fluorinated oil, one or more fluorinated additives to lowerevaporation potential, and one or more fluorinated co-surfactants, amongothers. The following formulations correspond to exemplary embodimentsof the nonaqueous phase of the present disclosure.

Oil Phase Formulation 1 (High surfactant)

-   -   HFE 7500 fluorinated oil    -   KRTOX-AS and/or KRYTOX-M (˜1-4% w/w)

Oil Phase Formulation 2

-   -   HFE 7500 fluorinated oil    -   KRYTOX-AS and/or KRYTOX-M (˜0.45-2.85% w/w)    -   Perfluorodecanol (˜0.009-2.25% w/w or ˜1.8% w/w)

Oil Phase Formulation 3

-   -   HFE 7500 fluorinated oil    -   KRYTOX-AS or KRYTOX-M (˜1.8% w/w)    -   Perfluorodecanol (˜0.18% w/w)

Oil Phase Formulation 4

-   -   FC-40 fluorinated oil    -   KRYTOX-AS and/or KRYTOX-M (˜0.45-2.85% w/w or ˜1.8% w/w)    -   Perfluorodecanol (˜0.009-2.25% w/w or ˜0.18% w/w)

Oil Phase Formulation 5

-   -   FC-43 fluorinated oil    -   KRYTOX-AS and/or KRYTOX-M (˜0.45-2.85% w/w or ˜1.8% w/w)    -   Perfluorodecanol (˜0.009-2.25% w/w or ˜0.18% w/w)

Oil Phase Formulation 6

-   -   FC-70 fluorinated oil    -   KRYTOX-AS and/or KRYTOX-M (˜0.45-2.85% w/w or ˜1.8% w/w)    -   Perfluorodecanol (˜0-2.25% w/w or ˜0.18% w/w)        Oil Phase Formulation 7 (Low surfactant)    -   HFE 7500 fluorinated oil solvent    -   KRTOX-AS and/or KRYTOX-M (greater than ˜0.01 to 0.5% w/w, ˜0.02        to 0.5% w/w, or ˜0.18% w/w)

V. FORMATION OF EMULSIONS

The aqueous and nonaqueous phases containing the components discussedabove may be provided (e.g., obtained and/or prepared), and thenutilized to form an emulsion.

An emulsion generally includes droplets of a dispersed phase (e.g., anaqueous phase) disposed in an immiscible continuous phase (e.g., anonaqueous phase such as an oil phase) that serves as a carrier fluidfor the droplets. Both the dispersed and continuous phases generally areat least predominantly liquid. The emulsion may be a water-in-oil (W/O)emulsion, an oil-in-water (0/W) emulsion or a multiple emulsion (e.g., aW/O/W or a W/O/W/O emulsion, among others).

Any suitable method and structure may be used to form the emulsion.Generally, energy input is needed to form the emulsion, such as shaking,stirring, sonicating, agitating, or otherwise homogenizing the emulsion.However, these approaches generally produce polydisperse emulsions, inwhich droplets exhibit a range of sizes, by substantially uncontrolledgeneration of droplets. Alternatively, monodisperse emulsions (with ahighly uniform size of droplets) may be created by controlled, serialdroplet generation with at least one droplet generator. In exemplaryembodiments, the droplet generator operates by microchannel flowfocusing to generate an emulsion of monodisperse droplets. Otherapproaches to and structures for droplet generation that may be suitableare described above in the documents listed above underCross-References, which are incorporated herein by reference,particularly U.S. Provisional Patent Application Ser. No. 61/341,218,filed Mar. 25, 2010; U.S. Provisional Patent Application Ser. No.61/409,106, filed Nov. 1, 2010; U.S. Provisional Patent Application Ser.No. 61/409,473, filed Nov. 2, 2010; U.S. Provisional Patent ApplicationSer. No. 61/410,769, filed Nov. 5, 2010; U.S. patent application Ser.No. 12/862,542, filed Aug. 24, 2010; and U.S. Patent ApplicationPublication No. 2010/0173394 A1, published Jul. 8, 2010.

A surfactant present in the aqueous phase may aid in the formation ofaqueous droplets within a nonaqueous phase. The surfactant may do so byphysically interacting with both the nonaqueous phase and the aqueousphase, stabilizing the interface between the phases, and forming aself-assembled interfacial layer. The surfactant generally increases thekinetic stability of the droplets significantly, substantially reducingcoalescence of the droplets, as well as reducing aggregation. Thedroplets (before transformation to capsules) may be relatively stable toshear forces created by fluid flow during fluidic manipulation. Forexample, the droplets may be stable to flow rates of at least 40 μL/minor 50 μL/min in a 100 μm or 200 μm channel using selected combinationsof nonaqueous and aqueous phase formulations.

The resulting droplets may have any suitable shape and size. Thedroplets may be spherical, when shape is not constrained. The averagediameter of the droplets may be about 1 to 500 μm, 5 to 500 μm, or 50 to500 μm, and the average volume of the droplets may be about 50 pL to 500nL, or 100 pL to 10 nL, among others.

The droplets may be formed and then collected as an emulsion in areservoir, such as vial, a test tube, a well of a plate, a chamber, orthe like. In some embodiments, the droplets may be collected as anemulsion in a PCR vial or plate, which is then thermocycled.Alternatively, or in addition, the droplets may be collected in areservoir and then transferred to a different container forthermocycling and/or may be manipulated and/or transported via fluidics,such as microfluidics.

VI. DROPLET TRANSFORMATION

Droplets may be transformed into capsules in which the droplets areencapsulated by a skin. Generally, droplets are transformed by heating.The droplets, the continuous phase, and/or the emulsion may be heated toa temperature sufficient for skin formation and for a time sufficient toproduce the skin. An inverse relationship may exist between thetemperature and the time sufficient for such a conversion to occur. Thatis, heating the droplets at a relatively low temperature may require alonger heating time than heating the droplets at a relatively highertemperature. However, skin formation may occur rapidly above a thresholdtemperature and much more slowly a few degrees below the thresholdtemperature. For example, skin formation may occur or be complete inless than about five minutes or less than about one minute when theemulsion is heated above the threshold temperature. In any event,transformation of droplets into capsules may decrease the solubility ofone or more skin-forming proteins (and/or other skin-formingmaterial(s)) in the aqueous phase (i.e., the dispersed phase orcontinuous phase), such that the proteins/materials become less soluble(e.g., substantially insoluble) in the aqueous phase. Accordingly, theskin may be substantially insoluble in the aqueous phase.

In some embodiments, the threshold temperature may correspond to thedenaturation temperature of a skin-forming protein in the aqueous phase.Accordingly, formation of the skin may be a consequence of proteindenaturation that occurs much more rapidly above the thresholdtemperature than below. As an example, BSA has been reported to denatureat about 50° C. to 55° C., and droplets incorporating BSA as askin-forming protein are induced to form a skin rapidly at about thesame temperature. Accordingly, use of another skin-forming protein witha different denaturation temperature may require heating to acorresponding different temperature before skin is formed.

Heating the droplets to a temperature above 55° C. may convert aself-assembled interfacial layer to an interfacial skin. The skin may becomposed of protein, or protein and surfactant, among others. In somecases, the droplets may be heated via thermal cycling, such as isperformed during PCR amplification. The thermal cycling profile mayinclude variations in temperature from about 4° C. to about 99° C. Thedroplets optionally may be heated via thermal cycling as a result oftransport of the droplets through a flow-based thermocycling system.Further aspects of an exemplary flow-based thermocycling system aredisclosed in U.S. Patent Application Publication No. 2010/0173394 A1,published Jul. 8, 2010, which is incorporated herein by reference.

VII. CAPSULES

Capsules enclose droplets in a skin, which can be visualizedmicroscopically when wrinkled, deformed, or damaged. The skin is a solidor semi-solid phase disposed interfacially, that is, near or at aninterface between each droplet boundary and the continuous phase.Accordingly, in contrast to the fluid interface present in standarddroplets, a decrease in capsule volume generally results in amicroscopically visible change in appearance of the skin. The skin maylose its smooth spherical geometry, as less tension is applied to theskin, and appear somewhat wrinkled or shriveled, reflecting asubstantial degree of solidity. For example, the presence (or absence)of a skin may be detected by spreading capsules/droplets on a microscopeslide, encouraging capsule/droplet shrinkage through evaporation, andthen observing the capsules/droplets under a microscope. Similarly, whenthe capsules are exposed to a non-optimal spacing fluid and/or aresubjected to excessive shear force, the skin may tear, leaving distinctopenings, with ragged edges, in the skin itself (e.g., see Example 3).

The skin may remain pliant and flexible, such that the capsules areviscoelastic. However, by suffering deformation and physical damage thatis readily observed visually (e.g., via microscope), the skins arerevealed to be at least substantially semi-solid or solid, and not afreely deformable liquid.

The enhanced stability of the capsule, relative to the original dropletformulation, is reflected in the stability of the capsule with respectto physical manipulation. The capsules may be transported, sorted, flowfocused, and dispensed with little or no damage to the capsule wall(i.e., the skin). In contrast to the precursor droplets, the capsulesmay be stable to fluidic processing operations that generate relativelyhigh shear. For example, the capsules may be stable in fluid flowing ata flow rate of up to at least about 200, 300 or 400 μL/min in a channelwith a diameter of about 125 μm or less or about 250 μm or less, amongothers, and/or may be stable flowing through 90-degree turns (as may beformed by valves).

The capsules may be used in any suitable manner. The capsules may becollected, manipulated, and/or sorted. They may be used in an assay orother biomedical application, or may be collected and stored. Thecapsules disclosed herein are typically stable with respect to storage,and may be stored at room temperature for one month or longer. Thecapsules may be stored at a wide range of temperatures, but preferablyfrom about 4° C. to about 40° C., among others.

A portion (e.g., a majority) of the continuous phase may be removedprior to heating of the droplets to create capsules. Where the majorityof the continuous phase has been removed, the resulting capsules mayoccupy a high fraction of the emulsion, resulting in a composition thatresembles a gel in some respects. The capsules may be densely packed insuch cases and, where the capsules originate from monodisperse droplets,may pack in a highly ordered arrangement.

Where the continuous phase is not removed prior to heating, theresulting composition typically resembles a fluid. Although the capsulesmay settle into a close-packed arrangement, agitation of the compositiontypically results in dispersion of the capsules in the continuous phase.

VIII. SPACING FLUID

A spacing fluid may be added to the emulsion. The spacing fluidgenerally is miscible with the current/original continuous phase of theemulsion and may have the same composition as, or a differentcomposition from, the current/original continuous phase. Accordingly,the spacing fluid may be nonaqueous or aqueous, based on the type ofemulsion to which the fluid is being added.

For use with a water-in-oil emulsion, the spacing fluid may include thesame base oil as the continuous phase or a different base oil. (A baseoil is the predominant or primary oil (or oils) in an oil (continuous)phase.) For example, the continuous phase may have a fluorinated oil asthe base oil, and the spacing fluid may have the same (or a different)fluorinated oil as its base oil.

In exemplary embodiments, the spacing fluid includes a differentsurfactant than the continuous phase, and/or substantially less totalsurfactant by weight than the continuous phase (e.g., at least about 2-,5-, 10-, or 100-fold less total surfactant, among others).Alternatively, or in addition, the spacing fluid may have no surfactantthat is present at a concentration above the critical micelleconcentration of the surfactant (which includes having at leastsubstantially no surfactant at all). Use of a concentration ofsurfactant below its critical micelle concentration may minimizeunwanted formation of new droplets, while providing a cleaning functionin a flow system. Also, with some emulsion formulations, use of the samesurfactant and approximately the same amount of surfactant in thespacing fluid as in the original continuous phase of the emulsion maycause capsules to shrink, shrivel, and/or rupture, which permits theskin to be visualized microscopically. Exemplary effects of distinctspacing fluids on capsule integrity are described below in Example 3.

In some examples, the continuous phase and the spacing fluid both maycontain ionic (primary) fluorosurfactants, or the continuous phase maycontain an ionic (primary) fluorosurfactant and the spacing fluid anonionic (primary) fluorosurfactant. If both contain an ionicfluorosurfactant, the primary fluorosurfactant concentration may beselected to be substantially lower in the spacing fluid than in thecontinuous phase. Otherwise, if the concentration of the ionicfluorosurfactant in the spacing fluid is too high, the ionicfluorosurfactant may draw water out of the capsules, causing them toshrink (which may wrinkle/tear the skin). If a non-ionicfluorosurfactant is used in the spacing fluid, then capsule shrinkagegenerally does not occur at either low or high concentrations ofsurfactant. However, shrinkage may depend on the purity of the non-ionicsurfactant. If a non-ionic surfactant is not 100% pure, ionic impuritiesmay exist (such as reactive precursors or reaction by-products). Athigher concentrations of an impure non-ionic surfactant, these ionicimpurities may reach a concentration high enough to cause damage to thecapsules (e.g., by withdrawal of water from the capsules to causeshrinkage or breakage). In any event, the nonionic fluorosurfactant maybe present in the spacing fluid at a substantially lower, about thesame, or a substantially higher concentration than the primary (ortotal) surfactant in the continuous phase (and/or an oil phase or oilcomposition used to form an emulsion).

The spacing fluid may be formulated according to the nonaqueous phasespresented above in Section IV. Additional exemplary formulations for aspacing fluid are as follows:

Spacing Fluid Formulation 1

-   -   HFE 7500, FC-40, FC-43, and/or FC-70 fluorinated oil    -   Perfluorinated alcohol (˜0-10% w/w or ˜0.18% w/w)    -   KRTOX-AS and/or KRYTOX-M (0-0.1%, 0-0.01%, or 0-0.001% w/w)

Spacing Fluid Formulation 2

-   -   HFE 7500, FC-40, FC-43, and/or FC-70 fluorinated oil    -   Pegylated-fluorosurfactant (0-0.1%, 0-0.01%, 0-0.001%, or        0.00001% w/w)

IX. CAPSULE AND DATA PROCESSING

The capsules of the present disclosure, once prepared, may beprocessing. Processing may include subjecting the capsules to anycondition or set of conditions under which at least one reaction ofinterest can occur (and/or is stopped), and for any suitable timeperiod. Accordingly, processing may include maintaining the temperatureof the capsules at or near a predefined set point, varying thetemperature of the capsules between two or more predefined set points(such as thermally cycling the capsules), exposing the capsules tolight, changing a pressure exerted on the capsules, applying an electricfield to the capsules, or any combination thereof, among others.

Signals may be detected from the capsules before, during, and/or afterprocessing. The signals may be detected optically, electrically,chemically, or a combination thereof, among others. The signals maycorrespond to at least one reaction of interest performed in thecapsules. In exemplary embodiments, the signals may be detected asfluorescence signals, which may include two or more types of signalsdistinguishable fluorescence signals.

Data corresponding to the detected signals may be processed. Dataprocessing may determining an assay result for each encapsulated assaymixture analyzed, which may be an analog or digital value.

X. EXAMPLES

The following examples describe selected aspects and embodiments of thepresent disclosure related to systems for making and using emulsions,particularly emulsions including droplets encapsulated by a skin. Theseexamples are intended for illustration and should not limit the entirescope of the present disclosure.

Example 1 Selective Removal of Continuous Phase from an Emulsion

This example describes an exemplary approach 80 to removing a continuousphase 82 selectively (relative to a dispersed phase 83) from an emulsion84, to increase the volume fraction occupied by droplets (or capsules)86; see FIG. 3. Emulsion 84 may be held by a reservoir 88 having a port90. The port may be formed near or at the bottom of the reservoir, ifdroplets 86 are buoyant in the continuous phase (as shown here), or maybe formed at a higher position of the reservoir if the droplets sink inthe continuous phase.

Buoyant droplets may move within the continuous phase over time towardthe top of the emulsion, in a process termed creaming, if the dropletsinitially have a more uniform distribution in the entire volume of thecontinuous phase. As a result, the droplets accumulate over time in anupper region of the continuous phase, to form a droplet layer 92 ofaggregated droplets that grows downward as buoyant droplets are added tothe bottom of the layer. A lower portion 94 of the emulsion becomesprogressively depleted of droplets as droplets migrate upward to layer92. The density difference between the aqueous phase and the continuousphase, and the viscosity of the continuous phase, determine how muchtime (e.g., seconds, minutes, or hours) is needed for most of thedroplets to join layer 92. In any event, a pressure drop may be createdbetween the top of the emulsion and port 90, to drive lower portion 94of continuous phase 82 selectively from the reservoir, indicated at 96,via port 90. For example, pressure, indicated at 98, may be applied tothe top of the emulsion, such as by regulating air pressure above theemulsion, or a vacuum may be applied to port 90 to draw the continuousphase through the port. In any event, a concentrated emulsion 99produced after selective removal of continuous phase 82 is shown in theright half of FIG. 3. In other embodiments, droplets that sink in thecontinuous phase may be driven through port 90 to separate droplets froman upper portion of the continuous phase.

Example 2 Overlaying an Emulsion

This example describes exemplary approaches to overlaying an emulsionwith liquid; see FIGS. 4 and 5. The use of an overlay may be suited foruse with an emulsion having buoyant droplets. In this case, buoyancycauses droplets to collect near the interface of the emulsion with air,which renders the droplets less protected by the continuous phase andmore vulnerable to evaporative loss. Also, the presence of an airinterface may render the emulsion more susceptible to heat-induceddamage to droplets (e.g., droplet breakage).

FIG. 4 illustrates an approach 110 to overlaying an emulsion 112 byusing an overlay emulsion 114. The overlay emulsion may or may not havesubstantially the same continuous phase 116 as underlying or primaryemulsion 112, but generally has a continuous phase that is miscible withthe continuous phase of the primary emulsion. The overlay emulsion mayinclude aqueous overlay droplets 118 that are distinguishable fromdroplets 120 in the underlying emulsion, such as based on size, adifference in detectability, or the like. For example, the overlaydroplets may be “blanks” or “dummy” droplets that lack the probe(s),label(s), and/or marker(s) that is present in the underlying (sample)droplets. These “blank” droplets can be configured to produce nointerfering signal during detection of assay signals. Alternatively, orin addition, the overlay droplets may include a visible dye 122 thatpermits the presence, position, and integrity of the overlay to be seenby eye without interfering with assay results. The visible dye may be acompound that is at least substantially nonfluorescent, such asbromphenol blue or Allura Red, among others. In any event, the overlaydroplets, even if the same density as the underlying sample droplets,will tend to remain above the sample droplets to form a distinct dropletlayer, as shown schematically in the figure. The overlay droplets mayserve as sacrificial droplets that are selectively damaged when theprimary emulsion and overlay emulsion are heated (such as during thermalcycling), because the overlay droplets are closer to the air interface.

Overlay droplets may be placed over sample droplets before any of thedroplets (overlay or sample) are transformed by heat to capsules.Alternatively, overlay droplets can be pre-treated with a heatincubation step to transform the droplets into overlay capsules beforethe overlay capsules are placed over sample droplets.

Furthermore, overlay droplets/capsules may be used as acontrol/calibrator for an assay system. For example, thesedroplets/capsules can be blank or configured to have an indicator (suchas a dye) to be used as a control/standard that provides informationabout an instrument or process that sample droplets/capsules are exposedto, including thermal cycling, detection of assay analytes in flow, andso on. In exemplary embodiments, dye-loaded overlay droplets/capsulescan be used as controls/standards for calibrating a detector ordetection method, such as providing signals that correspond toamplification-positive and/or amplification-negative droplets/capsules.Further aspects of using droplets as controls/calibrators are describedin U.S. Patent Application Publication No. 2010/0173394 A1, publishedJul. 8, 2010, which is incorporated herein by reference.

Blank capsules (or droplets) also can be used in a carrier fluid toclean, and/or assess the cleanliness of, an instrument within whichsample capsules (or droplets) are transported. The blank capsules can,for example, be introduced after a detection run of sample capsules, tofollow the sample capsules along a flow path through a detection regionof the instrument. The blank capsules may help to urge residual samplecapsules along the flow path, such as by mechanically shearing away suchresidual capsules. Accordingly, the use of blank capsules may help todetermine if unwanted sample capsules are still remaining in theinstrument (which could contaminate future runs of sample capsules).

Non-sample capsules (or droplets) therefore may be useful as part of anoverlay (to protect sample droplets/capsules from breakage and/ordegradation) or when used separately from sample droplets/capsules. Inother words, non-sample capsules can serve multiple functions in somecases.

FIG. 5 shows an approach 130 to covering an emulsion 132 with an overlayphase 134. The overlay phase is immiscible with underlying continuousphase 136 of the emulsion, and optionally immiscible with an aqueousphase 138 that composes droplets 140 of emulsion 132. The overlay phasealso may have a lower density than continuous phase 136 and, optionally,aqueous phase 138. For example, the underlying continuous phase may beformed at least predominantly of fluorinated oil and the overlay phasemay be formed at least predominantly of fluorophobic and/or lipophilicoil, such as a hydrocarbon oil (e.g., mineral oil). In some embodiments,overlay phase 134 may be aqueous, and phases 134 and 138 may have atleast substantially the same density and/or composition of salt, buffer,and/or surfactant, among others. In other words, the overlay phase mayhave a similar composition to the aqueous phase and/or may beosmotically balanced with respect to the aqueous phase. Accordingly, anaqueous overlay phase may include any combination of the componentsdescribed above in Section III, such as salt, buffer, protein,surfactant, a visible dye as described above, or any combinationthereof, among others.

Example 3 Spacing Fluids and Capsule Damage

This example describes exemplary spacing fluids with distinct effects oncapsule shape and integrity, and presents micrographs showing theseeffects; see FIGS. 6A, 6B, and 7A-D.

FIGS. 6A and 6B show a pair of micrographs of capsules from emulsionsthat have been exposed to a spacing fluid composed of a base oil withoutsurfactant (FIG. 6A) or with surfactant (FIG. 6B). Droplets weregenerated in HFE 7500 fluorinated oil with 1.8% w/w of Krytox-AS assurfactant. Excess continuous phase was removed, and then the dropletswere heated to form capsules. A spacing fluid was added to thecontinuous phase, which substantially increased the volume fraction ofthe continuous phase in the emulsion. The spacing fluid was either baseoil (HFE 7500) without surfactant (FIG. 6A) or with surfactant at thesame concentration as for droplet generation (1.8% w/w of Krytox-AS). Inother words, the spacing fluid had the same composition as the originalcontinuous phase (FIG. 6B) or was the same except that the surfactantwas omitted (FIG. 6A).

The absence or presence of surfactant in the spacing fluid can have adramatically different effect on capsule shape and integrity. FIG. 6Ashows that, without surfactant in the spacing fluid, capsule boundariesin FIG. 6A appear smooth and spherical. In other words, elimination ofsurfactant from the spacing fluid may have no negative effect on capsuleshape and integrity when the spacing fluid is added to the continuousphase. FIG. 6B shows that, with the same surfactant in the spacing fluidand continuous phase, and at the same concentration, the capsules appearto shrink and shrivel. The capsule boundaries become more irregular andwrinkled and less spherical.

FIG. 7 shows a set of micrographs of capsules treated as in FIG. 6B butviewed at higher magnification. Many of the capsules exhibit a skin thathas been damaged. The skin is often wrinkled and in many cases torn,with ragged edges visible.

Example 4 Selected Embodiments I

This example describes selected aspects and embodiments related topreparation and use of emulsions containing capsules, presented withoutlimitation as a series of numbered paragraphs.

1. A method of preparing stable capsules of an aqueous phase,comprising: (A) preparing an aqueous phase including a buffering agent,a first surfactant at a concentration of 0.1 to 1.0% by weight, and anon-specific binding protein at a concentration of 0.1 to 1.0% byweight; (B) preparing an organic phase including a fluorinated oil and afluorinated surfactant; (C) forming droplets of the aqueous phasedisposed within the organic phase, where each droplet has a defineddroplet boundary; and (D) heating the droplets sufficiently to convertthe droplet boundary to a semi-solid or solid skin that encapsulates theaqueous phase.

2. The method of paragraph 1, wherein the first surfactant is a nonionicsurfactant.

3. The method of paragraph 1, wherein the fluorinated surfactant is anonionic surfactant or an anionic surfactant.

4. The method of paragraph 1, wherein the fluorinated surfactant is acarboxylic acid-terminated perfluoropolyether, an ammonium salt of acarboxylic acid-terminated perfluoropolyether, or a morpholinoderivative of a carboxylic acid-terminated perfluoropolyether.

5. The method of paragraph 1, wherein the formed droplets arenon-coalescing, stable to flocculation, and stable with respect to flowrates of at least 40 μL/min.

6. A method of sample analysis within discrete encapsulated droplets,comprising: (A) preparing an aqueous phase including a PCR reactionbuffer, a magnesium salt, a nonionic surfactant that is a blockcopolymer of polypropylene oxide and polyethylene oxide at aconcentration of 0.1 to 1.0% by weight, a blocking protein at aconcentration of 0.1 to 1.0% by weight, a heat-stable polymerase, dNTPs,and a target nucleic acid; (B) preparing an organic phase including afluorinated oil and a fluorinated surfactant; (C) forming droplets ofthe aqueous phase disposed within the organic phase, where the dropletseach have a defined droplet boundary; (D) heating the dropletssufficiently to convert the droplet boundary to a semi-solid or solidskin that encapsulates the aqueous phase; and (E) detecting PCRamplification of a nucleic acid target within the droplets.

7. The method of paragraph 6, wherein detecting PCR amplificationincludes optically detecting PCR amplification.

8. The method of paragraph 7, wherein optically detecting PCRamplification includes detecting a fluorescently-labeled probe.

9. The method of paragraph 6, further comprising a step of manipulatingthe converted droplets by one or more of transporting, sorting,focusing, diluting, concentrating, and dispensing the converteddroplets.

10. The method of paragraph 9, wherein manipulating the converteddroplets includes manipulating using microfluidics.

Example 5 Selected Embodiments II

This example describes selected aspects and embodiments related toassays with emulsions that include capsules, presented withoutlimitation as a series of numbered paragraphs.

1. A method of performing an assay, comprising: (A) providing an aqueousphase including a sample and an effective concentration of one or moreskin-forming proteins; (B) forming an emulsion including droplets of theaqueous phase disposed in a nonaqueous continuous phase; (C) heating theemulsion to create an interfacial skin between each droplet and thecontinuous phase, to transform the droplets into capsules; and (D)collecting assay data related to the sample from the capsules.

2. The method of paragraph 1, wherein the step of heating includes astep of heating the emulsion to a temperature of at least about 55° C.

3. The method of paragraph 2, wherein the step of heating includes astep of heating the emulsion to a temperature above about 90° C.

4. The method of paragraph 1, further comprising a step of amplifying anucleic acid target in individual capsules.

5. The method of paragraph 4, further comprising a step of thermallycycling the emulsion to promote amplification of the nucleic acidtarget.

6. The method of paragraph 1, wherein the aqueous phase providedincludes the skin-forming proteins at a concentration of at least about0.01% by weight.

7. The method of paragraph 1, wherein the aqueous phase providedincludes the skin-forming proteins at a concentration of at least about0.03% by weight.

8. The method of paragraph 1, further comprising a step of providing anoil phase that is used as a nonaqueous continuous phase for the step offorming an emulsion, and wherein the oil phase includes anegatively-charged, fluorinated surfactant in the continuous phase.

9. The method of paragraph 1, wherein the fluorinated surfactant ispresent at a concentration of about 0.05% to 0.5% by weight.

10. The method of paragraph 1, wherein the capsules have a high packingdensity after the step of heating, and wherein the step of collectingdata includes a step of collecting data from capsules traveling seriallythrough a detection region.

11. The method of paragraph 1, wherein the step of collecting dataincludes a step of imaging capsules.

12. The method of paragraph 1, wherein the aqueous phase includes atleast one surfactant.

13. The method of paragraph 12, wherein the surfactant is present at aconcentration of about 0.01% to 5% by weight.

14. The method of paragraph 12, wherein the surfactant is present at aconcentration of about 0.1% to 1% by weight.

15. The method of paragraph 12, wherein the surfactant is present at aconcentration of about 0.5% by weight.

16. The method of paragraph 12, wherein the surfactant includes a blockcopolymer of polypropylene oxide and polyethylene oxide.

17. The method of paragraph 1, wherein the step of providing an aqueousphase includes a step of providing an aqueous phase including asurfactant and the skin-forming proteins, and wherein the surfactant isnot required for creation of the interfacial skin.

18. The method of paragraph 1, wherein the aqueous phase providedincludes the skin-forming proteins at a concentration of about 0.01% to10% by weight.

19. The method of paragraph 1, wherein the aqueous phase providedincludes the skin-forming proteins at a concentration of about 0.03% to3% by weight.

20. The method of paragraph 1, wherein the aqueous phase providedincludes the skin-forming proteins at a concentration of about 0.1% to1% by weight.

21. The method of paragraph 1, wherein the skin-forming proteins arerequired for formation of the interfacial skin.

22. The method of paragraph 1, wherein the skin-forming proteins areselected from the group consisting of serum albumin, casein, gelatin,and globulin.

23. The method of paragraph 22, wherein the skin-forming proteinsinclude bovine serum albumin (BSA).

24. The method of paragraph 1, wherein the step of forming an emulsionincludes a step of generating droplets having an average diameter ofabout 1 μm to 500 μm.

25. The method of paragraph 1, wherein the step of forming an emulsionincludes a step of serially generating droplets that are monodisperse.

26. The method of paragraph 1, further comprising a step of providing anoil phase including a fluorinated oil and at least one fluorinatedsurfactant, wherein the step of forming an emulsion includes a step ofgenerating droplets of the aqueous phase disposed in the oil phase.

27. The method of paragraph 26, wherein the at least one fluorinatedsurfactant includes a fluorinated polyether.

28. The method of paragraph 26, wherein the oil phase provided includesa fluorinated surfactant at a concentration of about 0.001% to 10% byweight.

29. The method of paragraph 26, wherein the oil phase provided includesa fluorinated surfactant at a concentration of about 0.05% to 2% byweight.

30. The method of paragraph 26, wherein the oil phase provided includesa fluorinated surfactant at a concentration of about 0.05% to 0.5% byweight.

31. The method of paragraph 1, wherein the sample includes a nucleicacid target, and wherein the step of collecting assay data includes astep of collecting assay data related to amplification of the nucleicacid target in capsules of the emulsion.

32. The method of paragraph 1, wherein the step of heating the emulsionis performed with the emulsion disposed in a container that is sealedwith a sealing member, further comprising a step of piercing the sealingmember after the step of heating.

33. The method of paragraph 1, wherein the step of heating the emulsionis performed with the emulsion disposed in a container, furthercomprising a step of disposing a tip of a fluid transport device in theemulsion and a step of moving capsules from the container into the fluidtransport device via the tip.

34. The method of paragraph 1, further comprising a step of driving flowof capsules through a detection region, wherein the step of collectingassay data is performed as the capsules travel through the detectionregion.

35. The method of paragraph 34, wherein the step of collecting assaydata includes a step of collecting assay data from individual capsulestraveling serially through the detection region.

36. The method of paragraph 1, wherein the step of collecting assay dataincludes a step of imaging capsules.

37. The method of paragraph 1, further comprising a step of driving flowof capsules in a continuous phase at a flow rate of at least about 100μL/min.

38. The method of paragraph 1, further comprising a step of selectivelyremoving a portion of the continuous phase after the step of forming anemulsion and before the step of heating the emulsion.

39. The method of paragraph 1, further comprising a step of placing anoverlay onto the emulsion before the step of heating the emulsion.

40. The method of paragraph 39, wherein the overlay includes dropletsdisposed in a continuous phase.

41. The method of paragraph 40, wherein the droplets of the overlay donot interfere with the step of collecting assay data.

42. The method of paragraph 40, wherein the overlay is an aqueous phaseor an oil phase that is immiscible with the continuous phase.

43. The method of paragraph 1, wherein the step of heating the emulsionis performed without an overlay on the emulsion such that the emulsionis in contact with air.

44. The method of paragraph 1, further comprising a step of adding aspacing fluid to the emulsion after the step of heating the emulsion,wherein the spacing fluid is miscible with the continuous phase.

45. The method of paragraph 44, further comprising a step of picking upcapsules of the emulsion with a fluid transport device after the step ofadding a spacing fluid.

46. The method of paragraph 44, wherein the spacing fluid contains nosurfactant that is present at a concentration substantially above thecritical micelle concentration of the surfactant.

47. The method of paragraph 44, further comprising a step of providingan oil phase that is used as a continuous phase for the step of formingan emulsion, wherein each of the oil phase and the spacing fluid has apercent by weight of surfactant, and wherein the percent by weight ofsurfactant in the oil phase is substantially higher than in the spacingfluid.

48. The method of paragraph 47, wherein the percent by weight ofsurfactant in the oil phase is at least about ten-fold higher than inthe spacing fluid.

49. The method of paragraph 44, further comprising a step of providingan oil phase that is used as a continuous phase for the step of formingan emulsion, wherein the oil phase includes an ionic surfactant and thespacing fluid includes a nonionic surfactant.

50. The method of paragraph 1, further comprising (1) a step of drivingflow of capsules in a carrier fluid along a flow path extending througha detection region, and (2) a step of adding a spacing fluid to thecarrier fluid in the flow path to space capsules before such capsulesreach the detection region.

51. The method of paragraph 50, wherein the spacing fluid contains nosurfactant that is present at a concentration substantially above thecritical micelle concentration of the surfactant.

52. The method of paragraph 50, further comprising a step of providingan oil phase that is used as a continuous phase for the step of formingan emulsion, wherein each of the oil phase and the spacing fluid has apercent by weight of surfactant, and wherein the percent by weight ofsurfactant in the oil phase is substantially higher than in the spacingfluid.

53. The method of paragraph 52, wherein the percent by weight ofsurfactant in the oil phase is at least about 100-fold higher than inthe spacing fluid.

54. The method of paragraph 50, further comprising a step of providingan oil phase that is used as a continuous phase for the step of formingan emulsion, wherein the oil phase includes an ionic surfactant and thespacing fluid includes a nonionic surfactant.

55. A method of performing an assay, comprising: (A) providing anaqueous phase including an effective concentration of one or moreskin-forming proteins; (B) providing an oil phase including afluorinated oil and at least one fluorinated surfactant; (C) forming anemulsion including droplets of the aqueous phase disposed in the oilphase; (D) transforming the droplets into capsules by creating aninterfacial skin between each droplet and the oil phase; (E) thermallycycling the capsules to amplify a nucleic acid target in individualcapsules; and (F) collecting amplification data from the capsules.

56. The method of paragraph 55, wherein the aqueous phase providedincludes the skin-forming proteins at a concentration of at least about0.01% by weight.

57. The method of paragraph 55, wherein the aqueous phase providedincludes the skin-forming proteins at a concentration of at least about0.03% by weight.

58. The method of paragraph 55, wherein the oil phase provided includesa fluorinated surfactant at a concentration of about 0.05% to 0.5% byweight.

59. The method of paragraph 55, wherein the capsules have a high packingdensity after the step of transforming, and wherein the step ofcollecting amplification data includes a step of collectingamplification data from capsules traveling serially through a detectionregion.

60. The method of paragraph 55, wherein the step of transformingincludes a step of heating the emulsion to a temperature of at leastabout 50° C.

61. A method of performing an assay, comprising: (A) providing an oilphase including a fluorinated oil and at least one ionic surfactant thatis fluorinated and negatively-charged; (B) forming an emulsion includingvolumes of an aqueous phase disposed in the oil phase; (C) heating theemulsion to a temperature of at least about 50° C.; (D) amplifying anucleic acid target in the volumes; and (E) collecting assay datarelated to amplification of the nucleic acid target in individualvolumes.

The disclosure set forth above may encompass multiple distinctinventions with independent utility. Although each of these inventionshas been disclosed in its preferred form(s), the specific embodimentsthereof as disclosed and illustrated herein are not to be considered ina limiting sense, because numerous variations are possible. The subjectmatter of the inventions includes all novel and nonobvious combinationsand subcombinations of the various elements, features, functions, and/orproperties disclosed herein. The following aims particularly point outcertain combinations and subcombinations regarded as novel andnonobvious. Inventions embodied in other combinations andsubcombinations of features, functions, elements, and/or properties maybe claimed in applications claiming priority from this or a relatedapplication. Such claims, whether directed to a different invention orto the same invention, and whether broader, narrower, equal, ordifferent in scope to the original claims, also are regarded as includedwithin the subject matter of the inventions of the present disclosure.

1. A method of generating a stabilized emulsion, comprising: providingan aqueous phase including an effective concentration of one or moreskin-forming proteins; forming an emulsion including droplets of adispersed phase disposed in a continuous phase, the aqueous phase beingthe continuous phase or the dispersed phase; and heating the emulsion tocreate an interfacial skin between each droplet and the continuousphase, to transform the droplets into capsules.
 2. The method of claim1, wherein the step of heating includes a step of heating the emulsionto a temperature of at least about 55° C.
 3. The method claim 1, furthercomprising a step of thermally cycling the capsules through multiplerounds of heating and cooling after the step of heating.
 4. The methodof claim 1, wherein the step of heating is part of a thermal cyclingprocess that includes multiple rounds of heating and cooling.
 5. Themethod of claim 1, further comprising a step of amplifying a nucleicacid target in one or more of the capsules.
 6. The method of claim 1,wherein the step of heating the emulsion includes a step of heating theemulsion to at least a threshold temperature.
 7. The method of claim 6,wherein the threshold temperature is a denaturation temperature of theskin-forming protein.
 8. The method of claim 1, wherein the aqueousphase provided includes the skin-forming proteins at a concentration ofat least about 0.01% by weight.
 9. The method of claim 1, wherein theaqueous phase provided includes the skin-forming proteins at aconcentration of at least about 0.03% by weight.
 10. The method of claim1, wherein the aqueous phase provided includes the skin-forming proteinsat a concentration of at least about 0.1% by weight.
 11. The method ofclaim 1, wherein the aqueous phase provided includes a surfactantincluding a block copolymer of polypropylene oxide and polyethyleneoxide.
 12. The method of claim 11, wherein the aqueous phase providedincludes the surfactant at a concentration of about 0.01% to 10% byweight.
 13. The method of claim 1, wherein the aqueous phase comprisesnucleic acid.
 14. The method of claim 1, wherein the step of providingan aqueous phase includes a step of providing an aqueous phase includinga surfactant and the skin-forming proteins, and wherein the surfactantis not required for creation of the interfacial skin.
 15. The method ofclaim 1, further comprising a step of providing an oil phase that isused as a continuous phase for the step of forming an emulsion, andwherein the oil phase includes a fluorinated surfactant that isnegatively charged.
 16. The method of claim 15, wherein the fluorinatedsurfactant is a carboxylate.
 17. The method of claim 15, wherein thecontinuous phase also includes a fluorinated alcohol.
 18. The method ofclaim 1, further comprising a step of providing an oil phase that isused as a continuous phase for the step of forming an emulsion, andwherein the oil phase includes at least one fluorinated surfactant at aconcentration of about 0.02% to 0.5% by weight.
 19. The method of claim1, wherein the skin-forming proteins are required for formation of theinterfacial skin.
 20. The method of claim 1, wherein the skin-formingproteins are selected from the group consisting of albumin, casein,gelatin, and globulin.
 21. The method of claim 1, wherein the step ofheating the emulsion is performed with the droplets disposed in athree-dimensional arrangement having a high packing density.
 22. Themethod of claim 1, further comprising a step of selectively removing aportion of the continuous phase after the step of forming an emulsionand before the step of heating the emulsion.
 23. The method of claim 1,wherein the step of forming an emulsion includes a step of generatingdroplets serially using a droplet generator.
 24. The method of claim 1,wherein the step of forming an emulsion includes a step of generatingmonodisperse droplets of the aqueous phase.
 25. The method of claim 24,wherein the step of generating monodisperse droplets includes a step ofgenerating monodisperse droplets having a diameter of about 1 μm to 500μm.
 26. The method of 1, wherein the capsules are buoyant in thecontinuous phase.
 27. The method of claim 1, further comprising a stepof placing an overlay onto the emulsion before the step of heating theemulsion.
 28. The method of claim 1, wherein the step of heating isperformed without an overlay on the emulsion such that the emulsion isin contact with air.
 29. A composition for generating a stabilizedemulsion, comprising: a continuous phase formed with an oil compositionincluding a fluorinated oil and at least one fluorinated surfactant; anda plurality of aqueous droplets disposed in the continuous phase andincluding an effective concentration of one or more skin-formingproteins, wherein heating the continuous phase and the aqueous dropletsabove a threshold temperature creates an interfacial skin between eachdroplet and the continuous phase, to transform the droplets intocapsules.
 30. The composition of claim 29, where the capsules provideindividual reaction mixtures for performing a reaction in the capsules.31. The composition of claim 30, wherein the capsules provide individualreaction mixtures for amplification of a nucleic acid target.
 32. Thecomposition of claim 29, wherein the aqueous droplets include the one ormore skin-forming proteins at a concentration of about 0.01% to 10% byweight.
 33. A stabilized emulsion, comprising: a continuous phase formedwith an oil composition including a fluorinated oil and at least onefluorinated surfactant; and a plurality of capsules disposed in thecontinuous phase, each capsule including a proteinaceous, interfacialskin encapsulating an aqueous phase.
 34. The stabilized emulsion ofclaim 33, wherein the aqueous phase provides a reaction mixture forperforming a reaction in individual capsules.
 35. The stabilizedemulsion of claim 34, wherein the aqueous phase provides a reactionmixture for performing amplification of a nucleic acid target inindividual capsules.
 36. The stabilized emulsion of claim 33, whereinthe proteinaceous skin includes at least one protein selected from thegroup consisting of albumin, casein, gelatin, and globulin.
 37. Thestabilized emulsion of claim 33, wherein the at least one fluorinatedsurfactant includes a first fluorinated surfactant that is negativelycharged and a second fluorinated surfactant that is an alcohol.
 38. Astabilized emulsion, comprising: a continuous phase formed with an oilcomposition including a fluorinated oil and at least one fluorinatedsurfactant; and a plurality of capsules disposed in the continuous phaseand each containing an aqueous phase, wherein the capsules are resistantto coalescence if disposed at a high packing density and incubated at90° C. for at least one minute.
 39. The stabilized emulsion of claim 38,wherein the capsules are resistant to coalescence if incubated at 90° C.for at least ten minutes.
 40. A method of emulsion preparation,comprising: generating aqueous droplets in a continuous phase thatincludes a fluorinated oil; transforming the droplets to capsules eachincluding an aqueous phase encapsulated by a proteinaceous, interfacialskin; and adding a spacing fluid to the continuous phase, the spacingfluid being miscible with the continuous phase and having a differentcomposition than the continuous phase.
 41. The method of claim 40,wherein the step of transforming is performed with the capsules disposedin a three-dimensional arrangement having a high packing density. 42.The method of claim 40, wherein the step of transforming includes a stepof heating the continuous phase.
 43. The method of claim 42, wherein thestep of heating the continuous phase includes a step of heating thecontinuous phase to a temperature of at least about 55° C.
 44. Themethod of claim 40, further comprising a step of selectively removing aportion of the continuous phase after the step of generating aqueousdroplets and before the step of transforming the droplets.
 45. Themethod of claim 40, further comprising a step of amplifying a nucleicacid target in individual capsules.
 46. The method of claim 45, furthercomprising a step of thermally cycling the capsules to promoteamplification of the nucleic acid target.
 47. The method of claim 40,further comprising a step of driving flow of capsules through adetection region, and a step of collecting assay data from capsules assuch capsules travel through the detection region.
 48. The method ofclaim 40, further comprising a step of imaging capsules to detect assaydata from capsules.
 49. The method of claim 40, wherein the spacingfluid contains no surfactant that is present at a concentrationsubstantially above the critical micelle concentration of thesurfactant.
 50. The method of claim 40, wherein the step of generatingis performed with an aqueous phase and an oil phase, wherein each of theoil phase and the spacing fluid has a percent by weight of surfactant,and wherein the percent by weight of surfactant in the oil phase issubstantially higher than in the spacing fluid.
 51. The method of claim50, wherein the percent by weight of surfactant in the oil phase is atleast about ten-fold higher than in the spacing fluid.
 52. The method ofclaim 40, wherein the step of generating is performed with an aqueousphase and an oil phase, wherein each of the oil phase and the spacingfluid has a percent by weight of ionic surfactant, and wherein thepercent by weight of ionic surfactant in the oil phase is substantiallyhigher than in the spacing fluid.
 53. The method of claim 40, whereinthe step of generating is performed with an aqueous phase and an oilphase, and wherein the oil phase includes an ionic surfactant, andwherein the spacing fluid includes a nonionic surfactant.
 54. The methodof claim 53, wherein each of the ionic surfactant and the nonionicsurfactant is a fluorinated polyether.
 55. The method of claim 53,wherein the nonionic surfactant has a concentration in the spacing fluidthat is about the same as or greater than a concentration of the ionicsurfactant in the oil phase.
 56. The method of claim 40, wherein thestep of generating is performed with an aqueous phase and an oil phase,and wherein each of the oil phase and the spacing fluid includes adifferent primary or exclusive surfactant.
 57. The method of claim 40,further comprising a step of selectively removing a portion of thecontinuous phase before the step of transforming.
 58. The method ofclaim 40, wherein the step of adding a spacing fluid does notsubstantially wrinkle or break the skin of more than a minority of thecapsules.
 59. A kit for emulsion preparation, comprising: an aqueousphase including an effective concentration of one or more skin-formingproteins; a nonaqueous continuous phase including a fluorinated oil andat least one fluorinated surfactant; and a droplet generator capable offorming an emulsion including droplets of the aqueous phase disposed inthe nonaqueous continuous phase, wherein heating the emulsion above athreshold temperature creates an interfacial skin between each dropletand the continuous phase, to transform the droplets into capsules. 60.The kit of claim 59, wherein the aqueous phase includes one or morereaction components for amplification of a nucleic acid target.
 61. Thekit of claim 59, wherein the step of heating the emulsion reduces asolubility of the one or more skin-forming proteins in the aqueousphase.
 62. A method of generating a stabilized emulsion, comprising:providing an oil phase including a fluorinated oil and at least oneionic surfactant that is fluorinated and negatively-charged; forming anemulsion including droplets of an aqueous phase disposed in the oilphase, wherein the aqueous phase provides a reaction mixture foramplification of a nucleic acid target; and heating the emulsion to atemperature of at least about 50° C.
 63. The method of claim 62, whereinthe at least one ionic surfactant is a fluorinated polyether.
 64. Themethod of claim 62, wherein the reaction mixture includes at least onemagnesium-dependent enzyme.
 65. A method of generating a stabilizedemulsion, comprising: providing an oil phase including a fluorinatedoil, a fluorinated alcohol, and a fluorinated surfactant; forming anemulsion including droplets of an aqueous phase disposed in the oilphase; and heating the emulsion to a temperature of at least about 50°C.
 66. The method of claim 65, wherein the fluorinated alcohol has nomore than two hydroxyl groups.
 67. The method of claim 65, wherein thefluorinated alcohol has no more than twenty carbons.
 68. The method ofclaim 65, wherein the fluorinated alcohol is perfluorodecanol.
 69. Themethod of claim 65, wherein the fluorinated surfactant is a fluorinatedpolyether.
 70. The method of claim 69, wherein the fluorinated polyetheris negatively charged.